Microorganisms and methods for improving product yields on methanol using acetyl-coa synthesis

ABSTRACT

The invention provides non-naturally occurring microbial organisms containing enzymatic pathways and/or metabolic modifications for enhancing carbon flux through acetyl-CoA. In some embodiments, the microbial organisms of the invention having such pathways also include pathways for generating reducing equivalents, formaldehyde fixation and/or formate assimilation. The enhanced carbon flux through acetyl-CoA, in combination with pathways for generating reducing equivalents, formaldehyde fixation and/or formate assimilation can, in some embodiments, be used for production of a bioderived compound. Accordingly, in some embodiments, the microbial organisms of the invention can include a pathway capable of producing a bioderived compound of the invention. The invention still further provides a bioderived compound produced by a microbial organism of the invention, culture medium having the bioderived compound of the invention, compositions having the bioderived compound of the invention, a biobased product comprising the bioderived compound of the invention, and a process for producing a bioderived compound of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/039,221, which is a United States National Stage Application under 35U.S.C. § 371 of International Patent Application No. PCT/US2014/067287,filed Nov. 25, 2014, which claims the benefit of priority of U.S.Provisional Application Ser. No. 61/945,056, filed Feb. 26, 2014, and61/911,414, filed Dec. 3, 2013, the entire contents of which are eachincorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to biosynthetic processes, andmore specifically to organisms having pathways for enhanced carbon fluxthrough acetyl-CoA.

1,3-butanediol (1,3-BDO) is a four carbon diol traditionally producedfrom acetylene via its hydration. The resulting acetaldehyde is thenconverted to 3-hydroxybutyraldehdye which is subsequently reduced toform 1,3-BDO. More recently, acetylene has been replaced by the lessexpensive ethylene as a source of acetaldehyde. 1,3-BDO is commonly usedas an organic solvent for food flavoring agents. It is also used as aco-monomer for polyurethane and polyester resins and is widely employedas a hypoglycemic agent. Optically active 1,3-BDO is a useful startingmaterial for the synthesis of biologically active compounds and liquidcrystals. Another use of 1,3-butanediol is that its dehydration affords1,3-butadiene (Ichikawa et al. Journal of Molecular Catalysis A—Chemical256:106-112 (2006); Ichikawa et al. Journal of Molecular CatalysisA—Chemical 231:181-189 (2005), which is useful in the manufacturesynthetic rubbers (e.g., tires), latex, and resins. The reliance onpetroleum based feedstocks for either acetylene or ethylene warrants thedevelopment of a renewable feedstock based route to 1,3-butanediol andto butadiene.

1,4-butanediol (1,4-BDO) is a valuable chemical for the production ofhigh performance polymers, solvents, and fine chemicals. It is the basisfor producing other high value chemicals such as tetrahydrofuran (THF)and gamma-butyrolactone (GBL). The value chain is comprised of threemain segments including: (1) polymers, (2) THF derivatives, and (3) GBLderivatives. In the case of polymers, 1,4-BDO is a comonomer forpolybutylene terephthalate (PBT) production. PBT is a medium performanceengineering thermoplastic used in automotive, electrical, water systems,and small appliance applications. Conversion to THF, and subsequently topolytetramethylene ether glycol (PTMEG), provides an intermediate usedto manufacture spandex products such as LYCRA® fibers. PTMEG is alsocombined with 1,4-BDO in the production of specialty polyester ethers(COPE). COPEs are high modulus elastomers with excellent mechanicalproperties and of resistance, allowing them to operate at high and lowtemperature extremes. PTMEG and 1,4-BDO also make thermoplasticpolyurethanes processed on standard thermoplastic extrusion,calendaring, and molding equipment, and are characterized by theiroutstanding toughness and abrasion resistance. The GBL produced from1,4-BDO provides the feedstock for making pyrrolidones, as well asserving the agrochemical market. The pyrrolidones are used as highperformance solvents for extraction processes of increasing use,including for example, in the electronics industry and in pharmaceuticalproduction.

1,4-BDO is produced by two main petrochemical routes with a fewadditional routes also in commercial operation. One route involvesreacting acetylene with formaldehyde, followed by hydrogenation. Morerecently 1,4-BDO processes involving butane or butadiene oxidation tomaleic anhydride, followed by hydrogenation have been introduced.1,4-BDO is used almost exclusively as an intermediate to synthesizeother chemicals and polymers.

Over 25 billion pounds of butadiene (1,3-butadiene, BD) are producedannually and is applied in the manufacture of polymers such as syntheticrubbers and ABS resins, and chemicals such as hexamethylenediamine and1,4-butanediol. For example, butadiene can be reacted with numerousother chemicals, such as other alkenes, e.g. styrene, to manufacturenumerous copolymers, e.g. acrylonitrile 1,3-butadiene styrene (ABS),styrene-1,3-butadiene (SBR) rubber, styrene-1,3-butadiene latex. Thesematerials are used in rubber, plastic, insulation, fiberglass, pipes,automobile and boat parts, food containers, and carpet backing Butadieneis typically produced as a by-product of the steam cracking process forconversion of petroleum feedstocks such as naphtha, liquefied petroleumgas, ethane or natural gas to ethylene and other olefins. The ability tomanufacture butadiene from alternative and/or renewable feedstocks wouldrepresent a major advance in the quest for more sustainable chemicalproduction processes.

Crotyl alcohol, also referred to as 2-buten-1-ol, is a valuable chemicalintermediate. It serves as a precursor to crotyl halides, esters, andethers, which in turn are chemical intermediates in the production ofmonomers, fine chemicals, agricultural chemicals, and pharmaceuticals.Exemplary fine chemical products include sorbic acid,trimethylhydroquinone, crotonic acid and 3-methoxybutanol. Crotylalcohol is also a precursor to 1,3-butadiene. Crotyl alcohol iscurrently produced exclusively from petroleum feedstocks. For exampleJapanese Patent 47-013009 and U.S. Pat. Nos. 3,090,815, 3,090,816, and3,542,883 describe a method of producing crotyl alcohol by isomerizationof 1,2-epoxybutane. The ability to manufacture crotyl alcohol fromalternative and/or renewable feedstocks would represent a major advancein the quest for more sustainable chemical production processes.

3-Buten-2-ol (also referenced to as methyl vinyl carbinol (MVC)) is anintermediate that can be used to produce butadiene. There aresignificant advantages to use of 3-buten-2-ol over 1,3-BDO because thereare fewer separation steps and only one dehydration step. 3-Buten-2-olcan also be used as a solvent, a monomer for polymer production, or aprecursor to fine chemicals. Accordingly, the ability to manufacture3-buten-2-ol from alternative and/or renewable feedstock would againpresent a significant advantage for sustainable chemical productionprocesses.

Adipic acid, a dicarboxylic acid, has a molecular weight of 146.14. Itcan be used is to produce nylon 6,6, a linear polyamide made bycondensing adipic acid with hexamethylenediamine. This is employed formanufacturing different kinds of fibers. Other uses of adipic acidinclude its use in plasticizers, unsaturated polyesters, and polyesterpolyols. Additional uses include for production of polyurethane,lubricant components, and as a food ingredient as a flavorant andgelling aid.

Historically, adipic acid was prepared from various fats usingoxidation. Some current processes for adipic acid synthesis rely on theoxidation of KA oil, a mixture of cyclohexanone, the ketone or Kcomponent, and cyclohexanol, the alcohol or A component, or of purecyclohexanol using an excess of strong nitric acid. There are severalvariations of this theme which differ in the routes for production of KAor cyclohexanol. For example, phenol is an alternative raw material inKA oil production, and the process for the synthesis of adipic acid fromphenol has been described. The other versions of this process tend touse oxidizing agents other than nitric acid, such as hydrogen peroxide,air or oxygen.

In addition to hexamethylenediamine (HMDA) being used in the productionof nylon-6,6 as described above, it is also utilized to makehexamethylene diisocyanate, a monomer feedstock used in the productionof polyurethane. The diamine also serves as a cross-linking agent inepoxy resins. HMDA is presently produced by the hydrogenation ofadiponitrile.

Caprolactam is an organic compound which is a lactam of 6-aminohexanoicacid (ε-aminohexanoic acid, 6-aminocaproic acid). It can alternativelybe considered cyclic amide of caproic acid. One use of caprolactam is asa monomer in the production of nylon-6. Caprolactam can be synthesizedfrom cyclohexanone via an oximation process using hydroxylammoniumsulfate followed by catalytic rearrangement using the Beckmannrearrangement process step.

Methylacrylic acid (MAA) is a key precursor of methyl methacrylate(MMA), a chemical intermediate with a global demand in excess of 4.5billion pounds per year, much of which is converted to polyacrylates.The conventional process for synthesizing methyl methacrylate (i.e., theacetone cyanohydrin route) involves the conversion of hydrogen cyanide(HCN) and acetone to acetone cyanohydrin which then undergoes acidassisted hydrolysis and esterification with methanol to give MAA.Difficulties in handling potentially deadly HCN along with the highcosts of byproduct disposal (1.2 tons of ammonium bisulfate are formedper ton of MAA) have sparked a great deal of research aimed at cleanerand more economical processes. As a starting material, MAA can easily beconverted into MAA via esterification with methanol.

Thus, there exists a need for the development of methods for effectivelyproducing commercial quantities of compounds such as fatty alcohols,1,3-butanediol, 1,4-butanediol, butadiene, crotyl alcohol, 3-buten-2-ol,adipate, 6-aminocaproate, caprolactam, hexamethylenediamine andmethacylic acid. The present invention satisfies this need and providesrelated advantages as well.

SUMMARY OF INVENTION

The invention provides non-naturally occurring microbial organismscontaining enzymatic pathways for enhancing carbon flux throughacetyl-CoA. In some embodiments, the microbial organisms of theinvention having such pathways also include pathways for generatingreducing equivalents, formaldehyde fixation and/or formate assimilation.The enhanced carbon flux through acetyl-CoA, in combination withpathways for generating reducing equivalents, formaldehyde fixationand/or formate assimilation can, in some embodiments, be used forproduction of a bioderived compound of the invention. Accordingly, insome embodiments, the microbial organisms of the invention can include apathway capable of producing a bioderived compound of the invention.Bioderived compounds of the invention include alcohols, glycols, organicacids, alkenes, dienes, organic amines, organic aldehydes, vitamins,nutraceuticals and pharmaceuticals. In some embodiments, the bioderivedcompound is 1,3-butanediol, crotyl alcohol, butadiene, 3-buten-2-ol,1,4-butanediol, adipate, 6-aminocaproate, caprolactam,hexamethylenediamine, methacrylic acid, 2-hydroxyisobutyric acid, or anintermediate thereto.

In some embodiments, a non-naturally occurring microbial organism of theinvention includes a methanol metabolic pathway as depicted in FIG. 2and an acetyl-CoA pathway as depicted in FIG. 1 or 3. In some aspectsthe microbial organism can further includes a formaldehyde fixationpathway and/or formate assimilation pathway as depicted in FIG. 1.Alternatively, in some embodiments, the non-naturally occurringmicrobial organism of the invention includes a formaldehyde fixationpathway as depicted in FIG. 1, a formate assimilation pathway asdepicted in FIG. 1, and/or an acetyl-CoA pathway as depicted in FIG. 1or 3.

In one aspect, the formaldehyde fixation pathway, formate assimilationpathway, and/or a methanol metabolic pathway present in the microbialorganisms of the invention enhances the availability of substratesand/or pathway intermediates, such as acetyl-CoA, and/or reducingequivalents, which can be utilized for bioderived compound productionthrough one or more bioderived compound pathways of the invention. Forexample, in some embodiments, a non-naturally occurring microbialorganism of the invention that includes a methanol metabolic pathway canenhance the availability of reducing equivalents in the presence ofmethanol and/or convert methanol to formaldehyde, a substrate for theformaldehyde fixation pathway. Likewise, a non-naturally occurringmicrobial organism of the invention having a formate assimilationpathway can reutilize formate to generate substrates and pathwayintermediates such as formaldehyde, pyruvate and/or acetyl-CoA. Inanother embodiment, a non-naturally occurring microbial organism of theinvention can include a pathway for producing acetyl-CoA and/orsuccinyl-CoA by a pathway depicted in FIG. 4. Such substrates,intermediates and reducing equivalents can be used to increase the yieldof a bioderived compound produced by the microbial organism.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism containing an acetyl-CoA pathway, a methanoloxidation pathway, a hydrogenase and/or a carbon monoxide dehydrogenase.Accordingly, in some embodiments, the invention provides a non-naturallyoccurring microbial organism having an acetyl-CoA pathway and at leastone exogenous nucleic acid encoding an acetyl-CoA pathway enzymeexpressed in a sufficient amount to produce or enhance carbon fluxthrough acetyl-CoA, wherein the acetyl-CoA pathway includes a pathwayshown in FIG. 3. In some embodiments, the invention provides anon-naturally occurring microbial organism having a methanol oxidationpathway enzyme expressed in a sufficient amount to produce formaldehydein the presence of methanol. An exemplary methanol oxidation pathwayenzyme is a methanol dehydrogenase as depicted in FIG. 1, Step A. Insome embodiments, the invention provides a non-naturally occurringmicrobial organism having a hydrogenase and/or a carbon monoxidedehydrogenase for generating reducing equivalents as depicted in FIGS. 2and 3.

The invention further provides non-naturally occurring microbialorganisms that have elevated or enhanced synthesis or yield ofacetyl-CoA (e.g. intracellular) or bioderived compound includingalcohols, diols, fatty acids, glycols, organic acids, alkenes, dienes,organic amines, organic aldehydes, vitamins, nutraceuticals andpharmaceuticals and methods of using those non-naturally occurringorganisms to produce such biosynthetic products. The enhanced synthesisof intracellular acetyl-CoA enables enhanced production of bioderivedcompounds for which acetyl-CoA is an intermediate and further, may havebeen rate-limiting.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having attenuation of one or more endogenous enzymes,which enhances carbon flux through acetyl-CoA, or a gene disruption ofone or more endogenous nucleic acids encoding such enzymes. For example,in some aspects, the endogenous enzyme can be selected from DHA kinase,methanol oxidase, PQQ-dependent methanol dehydrogenase, DHA synthase orany combination thereof.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having attenuation of one or more endogenous enzymesof a competing formaldehyde assimilation or dissimilation pathway or agene disruption of one or more endogenous nucleic acids encoding enzymesof a competing formaldehyde assimilation or dissimilation pathway.Examples of these endogenous enzymes are described herein.

The invention still further provides a bioderived compound produced by amicrobial organism of the invention, culture medium having thebioderived compound of the invention, compositions having the bioderivedcompound of the invention, a biobased product comprising the bioderivedcompound of the invention, and a process for producing a bioderivedcompound of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary metabolic pathways enabling the conversion ofCO2, formate, formaldehyde (Fald), methanol (MeOH), glycerol, xylose(XYL) and glucose (GLC) to acetyl-CoA (ACCOA) and exemplary endogenousenzyme targets for optional attenuation or disruption. The exemplarypathways can be combined with bioderived compound pathways, includingthe pathways depicted herein that utilize ACCOA, such as those depictedin FIGS. 4-10. The enzyme targets are indicated by arrows having A″markings. The endogenous enzyme targets include DHA kinase, methanoloxidase (AOX), PQQ-dependent methanol dehydrogenase (PQQ) and/or DHAsynthase. The enzymatic transformations shown are carried out by thefollowing enzymes: A) methanol dehydrogenase, B) 3-hexulose-6-phosphatesynthase, C) 6-phospho-3-hexuloisomerase, D) dihydroxyacetone synthase,E) formate reductase, F) formate ligase, formate transferase, or formatesynthetase, G) formyl-CoA reductase, H) formyltetmhydrofolatesynthetase, I) methenyltetrahydrofolate cyclohydrolase, J)methylenetetrahydrofolate dehydrogenase, K) spontaneous orformaldehyde-forming enzyme, L) glycine cleavage system, M) serinehydroxymethyltransferase, N) serine deaminase, O)methylenetetrahydrofolate reductase, P) acetyl-CoA synthase, Q) pyruvateformate lyase, R) pyruvate dehydrogenase, pyruvate ferredoxinoxidoreductase, or pyruvate:NADP+ oxidoreductase, S) formatedehydrogenase, T) fructose-6-phosphate phosphoketolase, U)xylulose-5-phosphate phosphoketolase, V) phosphotransacetylase, W)acetate kinase, X) acetyl-CoA transferase, synthetase, or ligase, Y)lower glycolysis including glyceraldehyde-3-phosphate dehydrogenase, Z)fructose-6-phosphate aldolase. See abbreviation list below for compoundnames.

FIG. 2 shows exemplary metabolic pathways that provide the extraction ofreducing equivalents from methanol, hydrogen, or carbon monoxide.Enzymes are: A) methanol methyltransferase, B) methylenetetrahydrofolatereductase, C) methylenetetrahydrofolate dehydrogenase, D)methenyltetrahydrofolate cyclohydrolase, E) formyltetrahydrofolatedeformylase, F) formyltetmhydrofolate synthetase, G) formate hydrogenlyase, H) hydrogenase, I) formate dehydrogenase, J) methanoldehydrogenase, K) spontaneous or formaldehyde activating enzyme, L)formaldehyde dehydrogenase, M) spontaneous orS-(hydroxymethyl)glutathione synthase, N) Glutathione-DependentFormaldehyde Dehydrogenase, O) S-formylglutathione hydrolase, P) carbonmonoxide dehydrogenase. See abbreviation list below for compound names.

FIG. 3 shows exemplary pathways which can be used to increase carbonflux through acetyl-CoA from carbohydrates when reducing equivalentsproduced by a methanol or hydrogen oxidation pathway provided herein areavailable. The enzymatic transformations shown are carried out by thefollowing enzymes: T) fructose-6-phosphate phosphoketolase, U)xylulose-5-phosphate phosphoketolase, V) phosphotransacetylase, W)acetate kinase, X) acetyl-CoA transferase, synthetase, or ligase, and H)hydrogenase. See abbreviation list below for compound names.

FIG. 4 shows exemplary metabolic pathways enabling the conversion of theglycolysis intermediate glyceraldehye-3-phosphate (G3P) to acetyl-CoA(ACCOA) and/or succinyl-CoA (SUCCOA). The enzymatic transformationsshown are carried out by the following enzymes: A) PEP carboxylase orPEP carboxykinase, B) malate dehydrogenase, C) fumarase, D) fumaratereductase, E) succinyl-CoA synthetase or transferase, F) pyruvate kinaseor PTS-dependent substrate import, G) pyruvate dehydrogenase, pyruvateformate lyase, or pyruvate:ferredoxin oxidoreductase, H) citratesynthase, I) aconitase, J) isocitrate dehydrogenase, K)alpha-ketoglutamte dehydrogenase, L) pyruvate carboxylase, M) malicenzyme, N) isocitrate lyase and malate synthase. See abbreviation listbelow for compound names.

FIG. 5 shows exemplary pathways enabling production of 1,3-butanediol,crotyl alcohol, and butadiene from acetyl-CoA. 1,3-butanediol, crotylalcohol, and butadiene production is carried out by the followingenzymes: A) acetyl-CoA carboxylase, B) an acetoacetyl-CoA synthase, C)an acetyl-CoA:acetyl-CoA acyltransferase, D) an acetoacetyl-CoAreductase (ketone reducing), E) a 3-hydroxybutyryl-CoA reductase(aldehyde forming), F) a 3-hydroxybutyryl-CoA hydrolase, transferase orsynthetase, G) a 3-hydroxybutyrate reductase, H) a3-hydroxybutyraldehyde reductase, I) chemical dehydration or FIG. 6, J)a 3-hydroxybutyryl-CoA dehydratase, K) a crotonyl-CoA reductase(aldehyde forming), L) a crotonyl-CoA hydrolase, transferase orsynthetase, M) a crotonate reductase, N) a crotonaldehyde reductase, O)a crotyl alcohol kinase, P) a 2-butenyl-4-phosphate kinase, Q) abutadiene synthase, R) a crotyl alcohol diphosphokinase, S) chemicaldehydration or a crotyl alcohol dehydratase, T) a butadiene synthase(monophosphate), T) a butadiene synthase (monophosphate), U) acrotonyl-CoA reductase (alcohol forming), and V) a 3-hydroxybutyryl-CoAreductase (alcohol forming). See abbreviation list below for compoundnames.

FIG. 6 shows exemplary pathways for converting 1,3-butanediol to3-buten-2-ol and/or butadiene. 3-Buten-2-ol and butadiene production iscarried out by the following enzymes: A. 1,3-butanediol kinase, B.3-hydroxybutyrylphosphate kinase, C. 3-hydroxybutyryldiphosphate lyase,D. 1,3-butanediol diphosphokinase, E. 1,3-butanediol dehydratase, F.3-hydroxybutyrylphosphate lyase, G. 3-buten-2-ol dehydratase or chemicaldehydration.

FIG. 7 shows exemplary pathways enabling production of 1,4-butanediolfrom succinyl-CoA. 1,4-Butanediol production is carried out by thefollowing enzymes: A) a succinyl-CoA transferase or a succinyl-CoAsynthetase, B) a succinyl-CoA reductase (aldehyde forming), C) a 4-HBdehydrogenase, D) a 4-HB kinase, E) a phosphotrans-4-hydroxybutyrylase,F) a 4-hydroxybutyryl-CoA reductase (aldehyde forming), G) a1,4-butanediol dehydrogenase, H) a succinate reductase, I) asuccinyl-CoA reductase (alcohol forming), J) a 4-hydroxybutyryl-CoAtransferase or 4-hydroxybutyryl-CoA synthetase, K) a 4-HB reductase, L)a 4-hydroxybutyryl-phosphate reductase, and M) a 4-hydroxybutyryl-CoAreductase (alcohol forming).

FIG. 8 shows exemplary pathways enabling production of adipate,6-aminocaproic acid, caprolactam, and hexamethylenediamine fromsuccinyl-CoA and acetyl-CoA. Adipate, 6-aminocaproic acid, caprolactam,and hexamethylenediamine production is carried out by the followingenzymes: A) 3-oxoadipyl-CoA thiolase, B) 3-oxoadipyl-CoA reductase, C)3-hydroxyadipyl-CoA dehydratase, D) 5-carboxy-2-pentenoyl-CoA reductase,E) adipyl-CoA reductase (aldehyde forming), F) 6-aminocaproatetransaminase or 6-aminocaproate dehydrogenase, G)6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoA synthase,H) amidohydrolase, I) spontaneous cyclization, J) 6-aminocaproyl-CoAreductase (aldehyde forming), K) HMDA transaminase or HMDAdehydrogenase, L) Adipyl-CoA hydrolase, adipyl-CoA ligase, adipyl-CoAtransferase, or phosphotransadipylase/adipate kinase.

FIG. 9 shows exemplary pathways enabling production of3-hydroxyisobutyrate and methacrylic acid from succinyl-CoA.3-Hydroxyisobutyrate and methacrylic acid production are carried out bythe following enzymes: A) Methylmalonyl-CoA mutase, B) Methylmalonyl-CoAepimerase, C) Methylmalonyl-CoA reductase (aldehyde forming), D)Methylmalonate semialdehyde reductase, E) 3-hydroxyisobutyratedehydratase, F) Methylmalonyl-CoA reductase (alcohol forming).

FIG. 10 shows exemplary pathways enabling production of2-hydroxyisobutyrate and methacrylic acid from acetyl-CoA.2-Hydroxyisobutyrate and methacrylic acid production are carried out bythe following enzymes: A) acetyl-CoA:acetyl-CoA acyltransferase, B)acetoacetyl-CoA reductase (ketone reducing), C) 3-hydroxybutyrl-CoAmutase, D) 2-hydroxyisobutyryl-CoA dehydratase, E) methacrylyl-CoAsynthetase, hydrolase, or transferase, F) 2-hydroxyisobutyryl-CoAsynthetase, hydrolase, or transferase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to metabolic and biosyntheticprocesses and microbial organisms capable of enhancing carbon fluxthrough acetyl-CoA. The invention disclosed herein is based, at least inpart, on non-naturally occurring microbial organisms capable ofsynthesizing a bioderived compound using an acetyl-CoA pathway, methanolmetabolic pathway, a formaldehyde fixation pathway, and/or a formateassimilation pathway in combination with a bioderived compound pathway.Additionally, in some embodiments, the non-naturally occurring microbialorganisms can further include a methanol oxidation pathway, ahydrogenase and/or a carbon monoxide dehydrogenase.

The following is a list of abbreviations and their correspondingcompound or composition names. These abbreviations, which are usedthroughout the disclosure and the figures. It is understood that one ofordinary skill in the art can readily identify thesecompounds/compositions by such nomenclature: MeOH or MEOH=methanol;Fald=formaldehyde; GLC=glucose; G6P=glucose-6-phosphate;H6P=hexulose-6-phosphate; F6P=fructose-6-phosphate; FDP=fructosediphosphate or fructose-1,6-diphosphate; DHA=dihydroxyacetone;DHAP=dihydroxyacetone phosphate; G3P=glyceraldehyde-3-phosphate;PYR=pyruvate; ACTP=acetyl-phosphate; ACCOA=acetyl-CoA;AACOA=acetoacetyl-CoA; MALCOA=malonyl-CoA; FTHF=formyltetrahydrofolate;THF=tetrahydrofolate; E4P=erythrose-4-phosphate:Xu5P=xyulose-5-phosphate; Ru5P=ribulose-5-phosphate;S7P=sedoheptulose-7-phosphate: R5P=ribose-5-phosphate; XYL=xylose;TCA=tricarboxylic acid; PEP=Phosphoenolpyruvate; OAA=Oxaloacetate;MAL=malate; CIT=citrate; ICIT=isocitrate; AKG=alpha-ketoglutarate;FUM=Fumamte; SUCC=Succinate; SUCCOA=Succinyl-CoA;3HBCOA=3-hydroxybutyryl-CoA; 3-HB=3-hydroxybutyrate;3HBALD=3-hydroxybutyraldehyde; 13BDO=1,3-butanediol;CROTCOA=crotonyl-CoA; CROT=crotonate; CROTALD=crotonaldehyde;CROTALC=crotyl alcohol; CROT-Pi=crotyl phosphate; CROT-PPi=crotyldiphosphate or 2-butenyl-4-diphosphate.

It is also understood that association of multiple steps in a pathwaycan be indicated by linking their step identifiers with or withoutspaces or punctuation; for example, the following are equivalent todescribe the 4-step pathway comprising Step W, Step X, Step Y and StepZ: steps WXYZ or W,X,Y,Z or W;X:X;Z or W-X-Y-Z. One of ordinary skillcan readily distinguish a single step designator of “AA” or “AB” or “AD”from a multiple step pathway description based on context and use in thedescription and figures herein.

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial organism's genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within an acetyl-CoAor bioderived compound biosynthetic pathway.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides, or functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” are intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

It is understood that when more than one exogenous nucleic acid isincluded in a microbial organism that the more than one exogenousnucleic acids refers to the referenced encoding nucleic acid orbiosynthetic activity, as discussed above. It is further understood, asdisclosed herein, that such more than one exogenous nucleic acids can beintroduced into the host microbial organism on separate nucleic acidmolecules, on polycistronic nucleic acid molecules, or a combinationthereof, and still be considered as more than one exogenous nucleicacid. For example, as disclosed herein a microbial organism can beengineered to express two or more exogenous nucleic acids encoding adesired pathway enzyme or protein. In the case where two exogenousnucleic acids encoding a desired activity are introduced into a hostmicrobial organism, it is understood that the two exogenous nucleicacids can be introduced as a single nucleic acid, for example, on asingle plasmid, on separate plasmids, can be integrated into the hostchromosome at a single site or multiple sites, and still be consideredas two exogenous nucleic acids. Similarly, it is understood that morethan two exogenous nucleic acids can be introduced into a host organismin any desired combination, for example, on a single plasmid, onseparate plasmids, can be integrated into the host chromosome at asingle site or multiple sites, and still be considered as two or moreexogenous nucleic acids, for example three exogenous nucleic acids.Thus, the number of referenced exogenous nucleic acids or biosyntheticactivities refers to the number of encoding nucleic acids or the numberof biosynthetic activities, not the number of separate nucleic acidsintroduced into the host organism.

As used herein, the phrase “enhance carbon flux” is intended to mean tointensify, increase, or further improve the extent or flow of metaboliccarbon through or to a desired pathway, pathway product, intermediate,or bioderived compound. The intensity, increase or improvement can berelative to a predetermined baseline of a pathway product, intermediateor bioderived compound. For example, an increased yield of acetyl-CoAcan be achieved per mole of methanol with a phosphoketolase enzymedescribed herein (see, e.g., FIG. 1) than in the absence of aphosphoketolase enzyme. Similarly, an increased yield of acetyl-CoA canbe achieved per mole of methanol with the formate assimilation enzymes(see, e.g., FIG. 1) than in the absence of the enzymes. Since anincreased yield of acetyl-CoA can be achieved, a higher yield ofacetyl-CoA derived compounds, such as 1,3-butanediol, crotyl alcohol,butadiene, 3-buten-2-ol, 1,4-butanediol, adipate, 6-aminocaproate,caprolactam, hexamethylenediamine, methacylic acid and2-hydroxyisobutyric acid the invention, can also be achieved.

As used herein, the term “gene disruption,” or grammatical equivalentsthereof, is intended to mean a genetic alteration that renders theencoded gene product inactive or attenuated. The genetic alteration canbe, for example, deletion of the entire gene, deletion of a regulatorysequence required for transcription or translation, deletion of aportion of the gene which results in a truncated gene product, or by anyof various mutation strategies that inactivate or attenuate the encodedgene product, for example, replacement of a gene's promoter with aweaker promoter, replacement or insertion of one or more amino acid ofthe encoded protein to reduce its activity, stability or concentration,or inactivation of a gene's transactivating factor such as a regulatoryprotein. One particularly useful method of gene disruption is completegene deletion because it reduces or eliminates the occurrence of geneticreversions in the non-naturally occurring microorganisms of theinvention. A gene disruption also includes a null mutation, which refersto a mutation within a gene or a region containing a gene that resultsin the gene not being transcribed into RNA and/or translated into afunctional gene product. Such a null mutation can arise from many typesof mutations including, for example, inactivating point mutations,deletion of a portion of a gene, entire gene deletions, or deletion ofchromosomal segments.

As used herein, the term “growth-coupled” when used in reference to theproduction of a biochemical product is intended to mean that thebiosynthesis of the referenced biochemical product is produced duringthe growth phase of a microorganism. In a particular embodiment, thegrowth-coupled production can be obligatory, meaning that thebiosynthesis of the referenced biochemical is an obligatory productproduced during the growth phase of a microorganism.

As used herein, the term “attenuate,” or grammatical equivalentsthereof, is intended to mean to weaken, reduce or diminish the activityor amount of an enzyme or protein. Attenuation of the activity or amountof an enzyme or protein can mimic complete disruption if the attenuationcauses the activity or amount to fall below a critical level requiredfor a given pathway to function. However, the attenuation of theactivity or amount of an enzyme or protein that mimics completedisruption for one pathway, can still be sufficient for a separatepathway to continue to function. For example, attenuation of anendogenous enzyme or protein can be sufficient to mimic the completedisruption of the same enzyme or protein for production of acetyl-CoA ora bioderived compound of the invention, but the remaining activity oramount of enzyme or protein can still be sufficient to maintain otherpathways, such as a pathway that is critical for the host microbialorganism to survive, reproduce or grow. Attenuation of an enzyme orprotein can also be weakening, reducing or diminishing the activity oramount of the enzyme or protein in an amount that is sufficient toincrease yield of acetyl-CoA or a bioderived compound of the invention,but does not necessarily mimic complete disruption of the enzyme orprotein.

As used herein, the term “bioderived” means derived from or synthesizedby a biological organism and can be considered a renewable resourcesince it can be generated by a biological organism. Such a biologicalorganism, in particular the microbial organisms of the invention, canutilize a variety of carbon sources described herein including feedstockor biomass, such as, sugars and carbohydrates obtained from anagricultural, plant, bacterial, or animal source. Alternatively, thebiological organism can utilize, for example, atmospheric carbon and/ormethanol as a carbon source.

As used herein, the term “biobased” means a product as described hereinthat is composed, in whole or in part, of a bioderived compound of theinvention. A biobased product is in contrast to a petroleum basedproduct, wherein such a product is derived from or synthesized frompetroleum or a petrochemical feedstock.

A “bioderived compound,” as used herein, refers to a target molecule orchemical that is derived from or synthesized by a biological organism.In the context of the present invention, engineered microbial organismsare used to produce a bioderived compound or intermediate thereof viaacetyl-CoA, including optionally further through acetoacetyl-CoA,malonyl-CoA and/or succinyl-CoA. Bioderived compounds of the inventioninclude, but are not limited to, alcohols, glycols, organic acids,alkenes, dienes, organic amines, organic aldehydes, vitamins,nutraceuticals and pharmaceuticals.

Alcohols of the invention, including biofuel alcohols, include primaryalcohols, secondary alcohols, diols and triols, preferably having C3 toC10 carbon atoms. Alcohols include n-propanol and isopropanol. Biofuelalcohols are preferably C3-C10 and include 1-Propanol, Isopropanol,1-Butanol, Isobutanol, 1-Pentanol, Isopentenol, 2-Methyl-1-butanol,3-Methyl-1-butanol, 1-Hexanol, 3-Methyl-1-pentanol, 1-Heptanol,4-Methyl-1-hexanol, and 5-Methyl-1-hexanol. Diols include propanediolsand butanediols, including 1,4 butanediol, 1,3-butanediol and2,3-butanediol. Fatty alcohols include C4-C27 fatty alcohols, includingC12-C18, especially C12-C14, including saturate or unsaturated linearfatty alcohols.

Further exemplary bioderived compounds of the invention include: (a)1,4-butanediol and intermediates thereto, such as 4-hydroxybutanoic acid(4-hydroxybutanoate, 4-hydroxybutyrate (4-HB); (b) butadiene(1,3-butadiene) and intermediates thereto, such as 1,4-butanediol,1,3-butanediol, 2,3-butanediol, crotyl alcohol, 3-buten-2-ol (methylvinyl carbinol) and 3-buten-1-ol; (c) 1,3-butanediol and intermediatesthereto, such as 3-hydroxybutyrate (3-HB), 2,4-pentadienoate, crotylalcohol or 3-buten-1-ol; (d) adipate, 6-aminocaproic acid (6-ACA),caprolactam, hexamethylenediamine (HMDA) and levulinic acid and theirintermediates, e.g. adipyl-CoA, 4-aminobutyryl-CoA; (e) methacrylic acid(2-methyl-2-propenoic acid) and its esters, such as methyl methacrylateand methyl methacrylate (known collectively as methacrylates),3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate and theirintermediates; (f) glycols, including 1,2-propanediol (propyleneglycol), 1,3-propanediol, glycerol, ethylene glycol, diethylene glycol,triethylene glycol, dipropylene glycol, tripropylene glycol, neopentylglycol and bisphenol A and their intermediates; (g) succinic acid andintermediates thereto; and (h) fatty alcohols, which are aliphaticcompounds containing one or more hydroxyl groups and a chain of 4 ormore carbon atoms, or fatty acids and fatty aldehydes thereof, which arepreferably C4-C27 carbon atoms. Fatty alcohols include saturated fattyalcohols, unsaturated fatty alcohols and linear saturated fattyalcohols. Examples fatty alcohols include butyl, pentyl, hexyl, heptyl,octyl, nonyl, decyl, undecyl and dodecyl alcohols, and theircorresponding oxidized derivatives, i.e. fatty aldehydes or fatty acidshaving the same number of carbon atoms. Preferred fatty alcohols, fattyaldehydes and fatty acids have C8 to C18 carbon atoms, especiallyC12-C18, C12-C14, and C16-C18, including C12, C13, C14, C15, C16, C17,and C18 carbon atoms. Preferred fatty alcohols include linearunsaturated fatty alcohols, such as dodecanol (C12; lauryl alcohol),tridecyl alcohol (C13; 1-tridecanol, tridecanol, isotridecanol),myristyl alcohol (C14; 1-tetradecanol), pentadecyl alcohol (C15;1-pentadecanol, pentadecanol), cetyl alcohol (C16; 1-hexadecanol),heptadecyl alcohol (C17; 1-n-heptadecanol, heptadecanol) and stearylalcohol (C18; 1-octadecanol) and unsaturated counterparts includingpalmitoleyl alcohol (C16 unsaturated; cis-9-hexadecen-1-ol), or theircorresponding fatty aldehydes or fatty acids.

1,4-Butanediol and intermediates thereto, such as 4-hydroxybutanoic acid(4-hydroxybutanoate, 4-hydroxybutyrate, 4-HB), are bioderived compoundsthat can be made via enzymatic pathways described herein and in thefollowing publications. Suitable bioderived compound pathways andenzymes, methods for screening and methods for isolating are found in:WO2008115840A2 published 25 Sep. 2008 entitled Compositions and Methodsfor the Biosynthesis of 1,4-Butanediol and Its Precursors;WO2010141780A1 published 9 Dec. 2010 entitled Process of SeparatingComponents of A Fermentation Broth; WO2010141920A2 published 9 Dec. 2010entitled Microorganisms for the Production of 1,4-Butanediol and RelatedMethods; WO2010030711A2 published 18 Mar. 2010 entitled Microorganismsfor the Production of 1,4-Butanediol; WO2010071697A1 published 24 Jun.2010 Microorganisms and Methods for Conversion of Syngas and OtherCarbon Sources to Useful Products; WO2009094485A1 published 30 Jul. 2009Methods and Organisms for Utilizing Synthesis Gas or Other GaseousCarbon Sources and Methanol; WO2009023493A1 published 19 Feb. 2009entitled Methods and Organisms for the Growth-Coupled Production of1,4-Butanediol; and WO2008115840A2 published 25 Sep. 2008 entitledCompositions and Methods for the Biosynthesis of 1,4-Butanediol and ItsPrecursors, which are all incorporated herein by reference.

Butadiene and intermediates thereto, such as 1,4-butanediol,2,3-butanediol, 1,3-butanediol, crotyl alcohol, 3-buten-2-ol (methylvinyl carbinol) and 3-buten-1-ol, are bioderived compounds that can bemade via enzymatic pathways described herein and in the followingpublications. In addition to direct fermentation to produce butadiene,1,3-butanediol, 1,4-butanediol, crotyl alcohol, 3-buten-2-ol (methylvinyl carbinol) or 3-buten-1-ol can be separated, purified (for anyuse), and then chemically dehydrated to butadiene by metal-basedcatalysis. Suitable bioderived compound pathways and enzymes, methodsfor screening and methods for isolating are found in: WO2011140171A2published 10 Nov. 2011 entitled Microorganisms and Methods for theBiosynthesis of Butadiene; WO2012018624A2 published 9 Feb. 2012 entitledMicroorganisms and Methods for the Biosynthesis of Aromatics,2,4-Pentadienoate and 1,3-Butadiene; WO2011140171A2 published 10 Nov.2011 entitled Microorganisms and Methods for the Biosynthesis ofButadiene; WO2013040383A1 published 21 Mar. 2013 entitled Microorganismsand Methods for Producing Alkenes; WO2012177710A1 published 27 Dec. 2012entitled Microorganisms for Producing Butadiene and Methods Relatedthereto; WO2012106516A1 published 9 Aug. 2012 entitled Microorganismsand Methods for the Biosynthesis of Butadiene; and WO2013028519A1published 28 Feb. 2013 entitled Microorganisms and Methods for Producing2,4-Pentadienoate, Butadiene, Propylene, 1,3-Butanediol and RelatedAlcohols, which are all incorporated herein by reference.

1,3-Butanediol and intermediates thereto, such as 2,4-pentadienoate,crotyl alcohol or 3-buten-1-ol, are bioderived compounds that can bemade via enzymatic pathways described herein and in the followingpublications. Suitable bioderived compound pathways and enzymes, methodsfor screening and methods for isolating are found in: WO2011071682A1published 16 Jun. 2011 entitled Methods and Organisms for ConvertingSynthesis Gas or Other Gaseous Carbon Sources and Methanol to1,3-Butanediol; WO2011031897A published 17 Mar. 2011 entitledMicroorganisms and Methods for the Co-Production of Isopropanol withPrimary Alcohols, Diols and Acids; WO2010127319A2 published 4 Nov. 2010entitled Organisms for the Production of 1,3-Butanediol; WO2013071226A1published 16 May 2013 entitled Eukaryotic Organisms and Methods forIncreasing the Availability of Cytosolic Acetyl-CoA, and for Producing1,3-Butanediol; WO2013028519A1 published 28 Feb. 2013 entitledMicroorganisms and Methods for Producing 2,4-Pentadienoate, Butadiene,Propylene, 1,3-Butanediol and Related Alcohols; WO2013036764A1 published14 Mar. 2013 entitled Eukaryotic Organisms and Methods for Producing1,3-Butanediol; WO2013012975A1 published 24 Jan. 2013 entitled Methodsfor Increasing Product Yields; and WO2012177619A2 published 27 Dec. 2012entitled Microorganisms for Producing 1,3-Butanediol and Methods RelatedThereto, which are all incorporated herein by reference.

Adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine andlevulinic acid, and their intermediates, e.g. 4-aminobutyryl-CoA, arebioderived compounds that can be made via enzymatic pathways describedherein and in the following publications. Suitable bioderived compoundpathways and enzymes, methods for screening and methods for isolatingare found in: WO2010129936A1 published 11 Nov. 2010 entitledMicroorganisms and Methods for the Biosynthesis of Adipate,Hexamethylenediamine and 6-Aminocaproic Acid; WO2013012975A1 published24 Jan. 2013 entitled Methods for Increasing Product Yields;WO2012177721A1 published 27 Dec. 2012 entitled Microorganisms forProducing 6-Aminocaproic Acid; WO2012099621A1 published 26 Jul. 2012entitled Methods for Increasing Product Yields; and WO2009151728published 17 Dec. 2009 entitled Microorganisms for the production ofadipic acid and other compounds, which are all incorporated herein byreference.

Methacrylic acid (2-methyl-2-propenoic acid) is used in the preparationof its esters, known collectively as methacrylates (e.g methylmethacrylate, which is used most notably in the manufacture ofpolymers). Methacrylate esters such as methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate and their intermediatesare bioderived compounds that can be made via enzymatic pathwaysdescribed herein and in the following publications. Suitable bioderivedcompound pathways and enzymes, methods for screening and methods forisolating are found in: WO2012135789A2 published 4 Oct. 2012 entitledMicroorganisms for Producing Methacrylic Acid and Methacrylate Estersand Methods Related Thereto; and WO2009135074A2 published 5 Nov. 2009entitled Microorganisms for the Production of Methacrylic Acid, whichare all incorporated herein by reference.

1,2-Propanediol (propylene glycol), n-propanol, 1,3-propanediol andglycerol, and their intermediates are bioderived compounds that can bemade via enzymatic pathways described herein and in the followingpublications. Suitable bioderived compound pathways and enzymes, methodsfor screening and methods for isolating are found in: WO2009111672A1published 9 Nov. 2009 entitled Primary Alcohol Producing Organisms;WO2011031897A1 17 Mar. 2011 entitled Microorganisms and Methods for theCo-Production of Isopropanol with Primary Alcohols, Diols and Acids;WO2012177599A2 published 27 Dec. 2012 entitled Microorganisms forProducing N-Propanol 1,3-Propanediol, 1,2-Propanediol or Glycerol andMethods Related Thereto, which are all incorporated herein byreferenced.

Succinic acid and intermediates thereto, which are useful to produceproducts including polymers (e.g. PBS), 1,4-butanediol, tetrahydrofuran,pyrrolidone, solvents, paints, deicers, plastics, fuel additives,fabrics, carpets, pigments, and detergents, are bioderived compoundsthat can be made via enzymatic pathways described herein and in thefollowing publication. Suitable bioderived compound pathways andenzymes, methods for screening and methods for isolating are found in:EP1937821A2 published 2 Jul. 2008 entitled Methods and Organisms for theGrowth-Coupled Production of Succinate, which is incorporated herein byreference.

Primary alcohols and fatty alcohols (also known as long chain alcohols),including fatty acids and fatty aldehydes thereof, and intermediatesthereto, are bioderived compounds that can be made via enzymaticpathways in the following publications. Suitable bioderived compoundpathways and enzymes, methods for screening and methods for isolatingare found in: WO2009111672 published 11 Sep. 2009 entitled PrimaryAlcohol Producing Organisms; WO2012177726 published 27 Dec. 2012entitled Microorganism for Producing Primary Alcohols and RelatedCompounds and Methods Related Thereto, which are all incorporated hereinby reference.

Further suitable bioderived compounds that the microbial organisms ofthe invention can be used to produce via acetyl-CoA, includingoptionally further through acetoacetyl-CoA and/or succinyl-CoA, areincluded in the invention. Exemplary well known bioderived compounds,their pathways and enzymes for production, methods for screening andmethods for isolating are found in the following patents andpublications: succinate (U.S. publication 2007/0111294, WO 2007/030830,WO 2013/003432), 3-hydroxypropionic acid (3-hydroxypropionate) (U.S.publication 2008/0199926, WO 2008/091627, U.S. publication2010/0021978), 1,4-butanediol (U.S. Pat. No. 8,067,214, WO 2008/115840,U.S. Pat. No. 7,947,483, WO 2009/023493, U.S. Pat. No. 7,858,350, WO2010/030711, U.S. publication 2011/0003355, WO 2010/141780, U.S. Pat.No. 8,129,169, WO 2010/141920, U.S. publication 2011/0201068, WO2011/031897, U.S. Pat. No. 8,377,666, WO 2011/047101, U.S. publication2011/0217742, WO 2011/066076, U.S. publication 2013/0034884, WO2012/177943), 4-hydroxybutanoic acid (4-hydroxybutanoate,4-hydroxybutyrate, 4-hydroxybutryate) (U.S. Pat. No. 8,067,214, WO2008/115840, U.S. Pat. No. 7,947,483, WO 2009/023493, U.S. Pat. No.7,858,350, WO 2010/030711, U.S. publication 2011/0003355, WO2010/141780, U.S. Pat. No. 8,129,155, WO 2010/071697), γ-butyrolactone(U.S. Pat. No. 8,067,214, WO 2008/115840, U.S. Pat. No. 7,947,483, WO2009/023493, U.S. Pat. No. 7,858,350, WO 2010/030711, U.S. publication2011/0003355, WO 2010/141780, U.S. publication 2011/0217742, WO2011/066076), 4-hydroxybutyryl-CoA (U.S. publication 2011/0003355, WO2010/141780, U.S. publication 2013/0034884, WO 2012/177943),4-hydroxybutanal (U.S. publication 2011/0003355, WO 2010/141780, U.S.publication 2013/0034884, WO 2012/177943), putrescine (U.S. publication2011/0003355, WO 2010/141780, U.S. publication 2013/0034884, WO2012/177943), Olefins (such as acrylic acid and acrylate ester) (U.S.Pat. No. 8,026,386, WO 2009/045637), acetyl-CoA (U.S. Pat. No.8,323,950, WO 2009/094485), methyl tetrahydrofolate (U.S. Pat. No.8,323,950, WO 2009/094485), ethanol (U.S. Pat. No. 8,129,155, WO2010/071697), isopropanol (U.S. Pat. No. 8,129,155, WO 2010/071697, U.S.publication 2010/0323418, WO 2010/127303, U.S. publication 2011/0201068,WO 2011/031897), n-butanol (U.S. Pat. No. 8,129,155, WO 2010/071697),isobutanol (U.S. Pat. No. 8,129,155, WO 2010/071697), n-propanol (U.S.publication 2011/0201068, WO 2011/031897), methylacrylic acid(methylacrylate) (U.S. publication 2011/0201068, WO 2011/031897),primary alcohol (U.S. Pat. No. 7,977,084, WO 2009/111672, WO2012/177726), long chain alcohol (U.S. Pat. No. 7,977,084, WO2009/111672, WO 2012/177726), adipate (adipic acid) (U.S. Pat. No.8,062,871, WO 2009/151728, U.S. Pat. No. 8,377,680, WO 2010/129936, WO2012/177721), 6-aminocaproate (6-aminocaproic acid) (U.S. Pat. No.8,062,871, WO 2009/151728, U.S. Pat. No. 8,377,680, WO 2010/129936, WO2012/177721), caprolactam (U.S. Pat. No. 8,062,871, WO 2009/151728, U.S.Pat. No. 8,377,680, WO 2010/129936, WO 2012/177721),hexamethylenediamine (U.S. Pat. No. 8,377,680, WO 2010/129936, WO2012/177721), levulinic acid (U.S. Pat. No. 8,377,680, WO 2010/129936),2-hydroxyisobutyric acid (2-hydroxyisobutyrate) (U.S. Pat. No.8,241,877, WO 2009/135074, U.S. publication 2013/0065279, WO2012/135789), 3-hydroxyisobutyric acid (3-hydroxyisobutyrate) (U.S. Pat.No. 8,241,877, WO 2009/135074, U.S. publication 2013/0065279, WO2012/135789), methacrylic acid (methacrylate) (U.S. Pat. No. 8,241,877,WO 2009/135074, U.S. publication 2013/0065279, WO 2012/135789),methacrylate ester (U.S. publication 2013/0065279, WO 2012/135789),fumarate (fumaric acid) (U.S. Pat. No. 8,129,154, WO 2009/155382),malate (malic acid) (U.S. Pat. No. 8,129,154, WO 2009/155382), acrylate(carboxylic acid) (U.S. Pat. No. 8,129,154, WO 2009/155382), methylethyl ketone (U.S. publication 2010/0184173, WO 2010/057022, U.S. Pat.No. 8,420,375, WO 2010/144746), 2-butanol (U.S. publication2010/0184173, WO 2010/057022, U.S. Pat. No. 8,420,375, WO 2010/144746),1,3-butanediol (U.S. publication 2010/0330635, WO 2010/127319, U.S.publication 2011/0201068, WO 2011/031897, U.S. Pat. No. 8,268,607, WO2011/071682, U.S. publication 2013/0109064, WO 2013/028519, U.S.publication 2013/0066035, WO 2013/036764), cyclohexanone (U.S.publication 2011/0014668, WO 2010/132845), terephthalate (terephthalicacid) (U.S. publication 2011/0124911, WO 2011/017560, U.S. publication2011/0207185, WO 2011/094131, U.S. publication 2012/0021478, WO2012/018624), muconate (muconic acid) (U.S. publication 2011/0124911, WO2011/017560), aniline (U.S. publication 2011/0097767, WO 2011/050326),p-toluate (p-toluic acid) (U.S. publication 2011/0207185, WO2011/094131, U.S. publication 2012/0021478, WO 2012/018624),(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (U.S. publication2011/0207185, WO 2011/094131, U.S. publication 2012/0021478, WO2012/018624), ethylene glycol (U.S. publication 2011/0312049, WO2011/130378, WO 2012/177983), propylene (U.S. publication 2011/0269204,WO 2011/137198, U.S. publication 2012/0329119, U.S. publication2013/0109064, WO 2013/028519), butadiene (1,3-butadiene) (U.S.publication 2011/0300597, WO 2011/140171, U.S. publication 2012/0021478,WO 2012/018624, U.S. publication 2012/0225466, WO 2012/106516, U.S.publication 2013/0011891, WO 2012/177710, U.S. publication 2013/0109064,WO 2013/028519), toluene (U.S. publication 2012/0021478, WO2012/018624), benzene (U.S. publication 2012/0021478, WO 2012/018624),(2-hydroxy-4-oxobutoxy)phosphonate (U.S. publication 2012/0021478, WO2012/018624), benzoate (benzoic acid) (U.S. publication 2012/0021478, WO2012/018624), styrene (U.S. publication 2012/0021478, WO 2012/018624),2,4-pentadienoate (U.S. publication 2012/0021478, WO 2012/018624, U.S.publication 2013/0109064, WO 2013/028519), 3-butene-1-ol (U.S.publication 2012/0021478, WO 2012/018624, U.S. publication 2013/0109064,WO 2013/028519), 3-buten-2-ol (U.S. publication 2013/0109064, WO2013/028519), 1,4-cyclohexanedimethanol (U.S. publication 2012/0156740,WO 2012/082978), crotyl alcohol (U.S. publication 2013/0011891, WO2012/177710, U.S. publication 2013/0109064, WO 2013/028519), alkene(U.S. publication 2013/0122563, WO 2013/040383, US 2011/0196180),hydroxyacid (WO 2012/109176), ketoacid (WO 2012/109176), wax esters (WO2007/136762) or caprolactone (U.S. publication 2013/0144029, WO2013/067432) pathway. The patents and patent application publicationslisted above that disclose bioderived compound pathways are hereinincorporated herein by reference.

Acetyl-CoA is the immediate precursor for the synthesis of bioderivedcompounds as shown in FIGS. 5-10. Phosphoketolase pathways make possiblesynthesis of acetyl-CoA without requiring decarboxylation of pyruvate(Bogorad et al, Nature, 2013, published online 29 Sep. 2013; UnitedStates Publication 2006-0040365), which thereby provides higher yieldsof bioderived compounds of the invention from carbohydrates and methanolthan the yields attainable without phosphoketolase enzymes.

For example, synthesis of an exemplary fatty alcohol, dodecanol, frommethanol using methanol dehydrogenase (step A of FIG. 1), a formaldehydeassimilation pathway (steps B, C, D of FIG. 1), the pentose phosphatepathway, and glycolysis can provide a maximum theoretical yield of0.0556 mole dodecanol/mole methanol.

18 CH₄O+9 O₂→C₁₂H₂₆O+23 H₂O+6 CO₂

However, if these pathways are combined with a phosphoketolase pathway(steps T, U, V, W, X of FIG. 1), a maximum theoretical yield of 0.0833mole dodecanol/mole methanol can be obtained if we assume that thepathway is not required to provide net generation of ATP for cell growthand maintenance requirements.

12 CH₄O→C₁₂H₂₆O+11 H₂O

ATP for energetic requirements can be synthesized, at the expense oflowering the maximum theoretical product yield, by oxidizing methanol toCO₂ using several combinations of enzymes depicted in FIG. 2,glycolysis, the TCA cycle, the pentose phosphate pathway, and oxidativephosphorylation.

Similarly, synthesis of isopropanol from methanol using methanoldehydrogenase (step A of FIG. 1), a formaldehyde assimilation pathway(steps B, C, D of FIG. 1), the pentose phosphate pathway and glycolysiscan provide a maximum theoretical yield of 0.1667 mole isopropanol/molemethanol.

6 CH₄O+4.5 O₂→C₃H₈O+8 H₂O+3 CO₂

However, if these pathways are applied in combination with aphosphoketolase pathway (steps T, U, V, W, X of FIG. 1), a maximumtheoretical yield of 0.250 mole isopropanol/mole methanol can beobtained.

4 CH₄O+1.5 O₂→C₃H₈O+4 H₂O+CO₂

The overall pathway is ATP and redox positive enabling synthesis of bothATP and NAD(P)H from conversion of MeOH to isopropanol. Additional ATPcan be synthesized, at the expense of lowering the maximum theoreticalproduct yield, by oxidizing methanol to CO₂ using several combinationsof enzymes depicted in FIG. 2, glycolysis, the TCA cycle, the pentosephosphate pathway, and oxidative phosphorylation.

Synthesis of several other products from methanol using methanoldehydrogenase (step A of FIG. 1), a formaldehyde assimilation pathway(steps B, C, D of FIG. 1), the pentose phosphate pathway and glycolysiscan provide the following maximum theoretical yield stoichiometries:

Product CH4O O2 NH3 Product H2O CO2 1,3-Butanediol 6.000 3.500 0.000 -->1.000 7.000 2.000 Crotyl Alcohol 6.000 3.500 0.000 --> 1.000 8.000 2.0003-Buten-2-ol 6.000 3.500 0.000 --> 1.000 8.000 2.000 Butadiene 6.0003.500 0.000 --> 1.000 9.000 2.000 2-Hydroxyisobutyrate 6.000 4.500 0.000--> 1.000 8.000 2.000 Methacrylate (via 2-hydroxyisobutyrate) 6.0004.500 0.000 --> 1.000 9.000 2.000 3-Hydroxyisobutyrate (oxidative TCAcycle) 6.000 4.500 0.000 --> 1.000 8.000 2.000 Methacrylate (via3-hydroxyisobutyrate) 6.000 4.500 0.000 --> 1.000 9.000 2.0001,4-Butanediol (oxidative TCA cycle) 6.000 3.500 0.000 --> 1.000 9.0002.000 Adipate (oxidative TCA cycle) 9.000 7.000 0.000 --> 1.000 13.0003.000 6-Aminocaproate (oxidative TCA cycle) 9.000 6.000 1.000 --> 1.00013.000 3.000 Caprolactam (via 6-aminocaproate) 9.000 6.000 1.000 -->1.000 14.000 3.000 Hexamethylenediamine (oxidative TCA cycle) 9.0005.000 2.000 --> 1.000 13.000 3.000

In the products marked “oxidative TCA cycle”, the maximum yieldstoichiometries assume that the reductive TCA cycle enzymes (e.g.,malate dehydrogenase, fumarase, fumarate reductase, and succinyl-CoAligase) are not utilized for product formation. Exclusive use of theoxidative TCA cycle for product formation can be advantageous forsuccinyl-CoA derived products such as 3-hydroxyisobutyrate,1,4-butanediol, adipate, 6-aminocaproate, and hexamethylenediaminebecause it enables all of the product pathway flux to originate fromalpha-ketoglutarate dehydrogenase—an irreversible enzyme in vivo.

However, if these pathways are applied in combination with aphosphoketolase pathway (steps T, U, V, W, X of FIG. 1), an increasedmaximum theoretical yield can be obtained as shown below:

Product CH4O O2 NH3 Product H2O CO2 1,3-Butanediol 4.000 0.500 0.000 -->1.000 3.000 0.000 Crotyl Alcohol 4.000 0.500 0.000 --> 1.000 4.000 0.0003-Buten-2-ol 4.000 0.500 0.000 --> 1.000 4.000 0.000 Butadiene 4.0000.500 0.000 --> 1.000 5.000 0.000 2-Hydroxyisobutyrate 4.000 1.500 0.000--> 1.000 4.000 0.000 Methacrylate (via 2-hydroxyisobutyrate) 4.0001.500 0.000 --> 1.000 5.000 0.000 3-Hydroxyisobutyrate (oxidative TCAcycle) 5.000 3.000 0.000 --> 1.000 6.000 1.000 Methacrylate (via3-hydroxyisobutyrate) 5.000 3.000 0.000 --> 1.000 7.000 1.0001,4-Butanediol (oxidative TCA cycle) 5.000 2.000 0.000 --> 1.000 5.0001.000 Adipate (oxidative TCA cycle) 7.000 4.000 0.000 --> 1.000 9.0001.000 6-Aminocaproate (oxidative TCA cycle) 7.000 3.000 1.000 --> 1.0009.000 1.000 Caprolactam (via 6-aminocaproate) 7.000 3.000 1.000 -->1.000 10.000 1.000 Hexamethylenediamine (oxidative TCA cycle) 7.0002.000 2.000 --> 1.000 9.000 1.000

The theoretical yield of bioderived compounds of the invention fromcarbohydrates including but not limited to glucose, glycerol, sucrose,fructose, xylose, arabinose, and galactose, can also be enhanced byphosphoketolase enzymes, particularly when reducing equivalents areprovided by an exogenous source such as hydrogen or methanol. This isbecause phosphoketolase enzymes provide acetyl-CoA synthesis with 100%carbon conversion efficiency (e.g., 3 acetyl-CoA's per glucose, 2.5acetyl-CoA's per xylose, 1.5 acetyl-CoA's per glycerol).

For example, synthesis of an exemplary fatty alcohol, dodecanol, fromglucose in the absence of phosphoketolase enzymes can reach a maximumtheoretical dodecanol yield of 0.3333 mole dodecanol/mole glucose.

3 C₆H₁₂O₆→C₁₂H₂₆O+5 H₂O+6 CO₂

However, if enzyme steps T, U, V, W, X of FIG. 1 are applied incombination with glycolysis, the pentose phosphate pathway, and anexternal redox source (e.g., methanol, hydrogen) using the pathwaysshown in FIG. 2, the maximum theoretical yield can be increased to0.5000 mole dodecanol/mole glucose.

2 C₆H₁₂O₆+4 CH₄O→C₁₂H₂₆O+7 H₂O+4 CO₂

This assumes that the pathway is not required to provide net generationof ATP for cell growth and maintenance requirements. ATP for energeticrequirements can be synthesized by oxidizing additional methanol to CO₂using several combinations of enzymes depicted in FIG. 2.

Similarly, synthesis of isopropanol from glucose in the absence ofphosphoketolase enzymes can achieve a maximum theoretical isopropanolyield of 1.000 mole isopropanol/mole glucose.

C₆H₁₂O₆+1.5 O₂→C₃H₈O+2 H₂O+3 CO₂

However, if enzyme steps T, U, V, W, X of FIG. 1 are applied incombination with glycolysis and the pentose phosphate pathway, themaximum theoretical yield can be increased to 1.333 moleisopropanol/mole glucose.

C₆H₁₂O₆→1.333 C₃H₈O+0.667 H₂O+2 CO₂

If enzyme steps T, U, V, W, X of FIG. 1 are applied in combination withglycolysis, the pentose phosphate pathway, and external redox source(e.g., methanol, hydrogen) using the pathways shown in FIG. 2, themaximum theoretical yield can be increased to 1.500 moleisopropanol/mole glucose.

C₆H₁₂O₆+0.5 CH₄O→1.5 C₃H₈O+H₂O+2 CO₂

In the absence of phosphoketolase activity, synthesis of several otherproducts from a carbohydrate source (e.g., glucose) can provide thefollowing maximum theoretical yield stoichiometries using glycolysis,pentose phosphate pathway, and TCA cycle reactions to build the pathwayprecursors.

Product C6H12O6 O2 NH3 Product H2O CO2 1,3-Butanediol 1.000 0.500 0.000→ 1.000 1.000 2.000 Crotyl Alcohol 1.000 0.500 0.000 → 1.000 2.000 2.0003-Buten-2-ol 1.000 0.500 0.000 → 1.000 2.000 2.000 Butadiene 1.000 0.5000.000 → 1.000 3.000 2.000 2-Hydroxyisobutyrate 1.000 1.500 0.000 → 1.0002.000 2.000 Methacrylate (via2-hydroxyisobutyrate) 1.000 1.500 0.000 →1.000 3.000 2.000 3-Hydroxyisobutyrate (oxidative TCA cycle) 1.000 1.5000.000 → 1.000 2.000 2.000 Methacrylate (via 3-hydroxyisobutyrate) 1.0001.500 0.000 → 1.000 3.000 2.000 1,4-Butanediol (oxidative TCA cycle)1.000 0.500 0.000 → 1.000 1.000 2.000 Adipate (oxidative TCA cycle)1.000 1.667 0.000 → 0.667 2.667 2.000 6-Aminocaproate (oxidative TCAcycle) 1.000 1.000 0.667 → 0.667 2.667 2.000 Caprolactam (via6-aminocaproate) 1.000 1.000 0.667 → 0.667 3.333 2.000Hexamethylenediamine (oxidative TCA cycle) 1.000 0.333 1.333 → 0.6672.667 2.000

In the products marked “oxidative TCA cycle”, the maximum yieldstoichiometries assume that the TCA cycle enzymes (e.g., malatedehydrogenase, fumarase, fumarase reductase, and succinyl-CoA ligase)are not utilized for product formation in the reductive direction.Exclusive use of the oxidative TCA cycle for product formation can beadvantageous for succinyl-CoA derived products such as3-hydroxyisobutyrate, 1,4-butanediol, adipate, 6-aminocaproate, andhexamethylenediamine because it enables all of the product pathway fluxto originate from alpha-ketoglutarate dehydrogenase—an irreversibleenzyme in vivo.

Notably, when these product pathways are applied in combination with aphosphoketolase pathway (steps T, U, V, W, X of FIG. 1), an increasedmaximum theoretical yield can be obtained as shown below:

Product C6H12O6 O2 NH3 Product H2O CO2 1,3-Butanediol 1.000 0.000 0.000→ 1.091 0.545 1.636 Crotyl Alcohol 1.000 0.000 0.000 → 1.091 1.636 1.6363-Buten-2-ol 1.000 0.000 0.000 → 1.091 1.636 1.636 Butadiene 1.000 0.1070.000 → 1.071 2.786 1.714 2-Hydroxyisobutyrate 1.000 0.014 0.000 → 1.3300.679 0.679 Methacrylate (via2-hydroxyisobutyrate) 1.000 0.014 0.000 →1.330 2.009 0.679 3-Hydroxyisobutyrate (oxidative TCA cycle) 1.000 0.6000.000 → 1.200 1.200 1.200 Methacrylate (via 3-hydroxyisobutyrate) 1.0000.600 0.000 → 1.200 2.400 1.200 1,4-Butanediol (oxidative TCA cycle)1.000 0.124 0.000 → 1.068 0.658 1.727 Adipate (oxidative TCA cycle)1.000 0.429 0.000 → 0.857 1.714 0.857 6-Aminocaproate (oxidative TCAcycle) 1.000 0.000 0.800 → 0.800 2.000 1.200 Caprolactam (via6-aminocaproate) 1.000 0.000 0.800 → 0.800 2.800 1.200Hexamethylenediamine (oxidative TCA cycle) 1.000 0.064 1.397 → 0.6982.508 1.810

As with glucose, a similar yield increase can occur with use of aphosphoketolase enzyme on other carbohydrates such as glycerol, sucrose,fructose, xylose, arabinose and galactose.

Also provided herein are methanol metabolic pathways and a methanoloxidation pathway to improve that availability of reducing equivalentsand/or substrates for production of a compound of the invention. Becausemethanol is a relatively inexpensive organic feedstock that can be usedas a redox, energy, and carbon source for the production of bioderivedcompounds of the invention, and their intermediates, it is a desirablesubstrate for the non-naturally occurring microbial organisms of theinvention. Employing one or more methanol metabolic enzymes as describedherein, for example as shown in FIGS. 1 and 2, methanol can entercentral metabolism in most production hosts by employing methanoldehydrogenase (FIG. 1, step A) along with a pathway for formaldehydeassimilation. One exemplary formaldehyde assimilation pathway that canutilize formaldehyde produced from the oxidation of methanol is shown inFIG. 1, which involves condensation of formaldehyde andD-ribulose-5-phosphate to form hexulose-6-phosphate (H6P) byhexulose-6-phosphate synthase (FIG. 1, step B). The enzyme can use Mg²⁺or Mn²⁺ for maximal activity, although other metal ions are useful, andeven non-metal-ion-dependent mechanisms are contemplated. H6P isconverted into fructose-6-phosphate by 6-phospho-3-hexuloisomerase (FIG.1, step C). Another exemplary pathway that involves the detoxificationand assimilation of formaldehyde produced from the oxidation of methanolproceeds through dihydroxyacetone. Dihydroxyacetone synthase (FIG. 1,step D) is a transketolase that first transfers a glycoaldehyde groupfrom xylulose-5-phosphate to formaldehyde, resulting in the formation ofdihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P), which is anintermediate in glycolysis. The DHA obtained from DHA synthase can bethen further phosphorylated to form DHA phosphate by a DHA kinase. DHAPcan be assimilated into glycolysis, e.g. via isomerization to G3P, andseveral other pathways. Alternatively, DHA and G3P can be converted byfructose-6-phosphate aldolase to form fructose-6-phosphate (F6P) (FIG.1, step Z).

By combining the pathways for methanol oxidation (FIG. 1, step A) andformaldehyde fixation (FIG. 1, Steps B and C or Step D), molar yields of0.333 mol acetyl-CoA/mol methanol can be achieved for production of abioderived compound and their intermediates. The following maximumtheoretical yield stoichiometries for a fatty alcohol (e.g., a C12), afatty acid (e.g., a C12), a fatty aldehyde (e.g., a C12), isopropanol,1,3-butanediol, crotyl alcohol, butadiene, 3-buten-2-ol, 1,4-butanediol,adipate, 6-aminocaproate, caprolactam, hexamethylenediamine, methacylicacid and 2-hydroxyisobutyric acid are thus made possible by combiningthe steps for methanol oxidation, formaldehyde fixation, and productsynthesis.

18 CH₄O+9 O₂→C₁₂H₂₆O+6 CO₂+23 H₂O  (Fatty Alcohol on MeOH)

18 CH₄O+10 O₂→C₁₂H₂₄O₂+6 CO₂+24 H₂O  (Fatty Acid on MeOH)

18 CH₄O+9.5 O₂→C₁₂H₂₄O+6 CO₂+24 H₂O  (Fatty Aldehyde on MeOH)

6 CH₄O+4.5 O₂→C3H₈O+3 CO₂+8 H₂O  (Isopropanol on MeOH)

Additional stoichiometries are shown in the table below:

Product CH4O O2 NH3 Product H2O CO2 1,3-Butanediol 6.000 3.500 0.000 -->1.000 7.000 2.000 Crotyl Alcohol 6.000 3.500 0.000 --> 1.000 8.000 2.0003-Buten-2-ol 6.000 3.500 0.000 --> 1.000 8.000 2.000 Butadiene 6.0003.500 0.000 --> 1.000 9.000 2.000 2-Hydroxyisobutyrate 6.000 4.500 0.000--> 1.000 8.000 2.000 Methacrylate (via 2-hydroxyisobutyrate) 6.0004.500 0.000 --> 1.000 9.000 2.000 3-Hydroxyisobutyrate (oxidative TCAcycle) 6.000 4.500 0.000 --> 1.000 8.000 2.000 Methacrylate (via3-hydroxyisobutyrate) 6.000 4.500 0.000 --> 1.000 9.000 2.0001,4-Butanediol (oxidative TCA cycle) 6.000 3.500 0.000 --> 1.000 9.0002.000 Adipate (oxidative TCA cycle) 9.000 7.000 0.000 --> 1.000 13.0003.000 6-Aminocaproate (oxidative TCA cycle) 9.000 6.000 1.000 --> 1.00013.000 3.000 Caprolactam (via 6-aminocaproate) 9.000 6.000 1.000 -->1.000 14.000 3.000 Hexamethylenediamine (oxidative TCA cycle) 9.0005.000 2.000 --> 1.000 13.000 3.000

In the products marked “oxidative TCA cycle”, the maximum yieldstoichiometries assume that the reductive TCA cycle enzymes (e.g.,malate dehydrogenase, fumarase, fumarate reductase, and succinyl-CoAligase) are not utilized for product formation. Exclusive use of theoxidative TCA cycle for product formation can be advantageous forsuccinyl-CoA derived products such as 3-hydroxyisobutyrate,1,4-butanediol, adipate, 6-aminocaproate, and hexamethylenediaminebecause it enables all of the product pathway flux to originate fromalpha-ketoglutarate dehydrogenase—an irreversible enzyme in vivo.

The yield on several substrates, including methanol, can be furtherincreased by capturing some of the carbon lost from the conversion ofpathway intermediates, e.g. pyruvate to acetyl-CoA, using one of theformate reutilization pathways shown in FIG. 1. For example, the CO₂generated by conversion of pyruvate to acetyl-CoA (FIG. 1, step R) canbe converted to formate via formate dehydrogenase (FIG. 1, step S).Alternatively, pyruvate formate lyase, which forms formate directlyinstead of CO₂, can be used to convert pyruvate to acetyl-CoA (FIG. 1,step Q). Formate can be converted to formaldehyde by using: 1) formatereductase (FIG. 1, step E), 2) a formyl-CoA synthetase, transferase, orligase along with formyl-CoA reductase (FIG. 1, steps F-G), or 3)formyltetrahydrofolate synthetase, methenyltetrahydrofolatecyclohydrolase, methylenetetrahydrofolate dehydrogenase, andformaldehyde-forming enzyme (FIG. 1, steps H-I-J-K). Conversion ofmethylene-THF to formaldehyde alternatively will occur spontaneously.Alternatively, formate can be reutilized by converting it to pyruvate oracetyl-CoA using FIG. 1, steps H-I-J-L-M-N or FIG. 1, steps H-I-J-O-P,respectively. Formate reutilization is also useful when formate is anexternal carbon source. For example, formate can be obtained fromorganocatalytic, electrochemical, or photoelectrochemical conversion ofCO2 to formate. An alternative source of methanol for use in the presentmethods is organocatalytic, electrochemical, or photoelectrochemicalconversion of CO₂ to methanol.

By combining the pathways for methanol oxidation (FIG. 1, step A),formaldehyde fixation (FIG. 1, Steps B and C or Step D), and formatereutilization, molar yields as high as 0.500 mol acetyl-CoA/mol methanolcan be achieved for production of a bioderived compound and theirintermediates. Thus, for example, the following maximum theoreticalyield stoichiometries for a fatty alcohol (e.g., a C12), a fatty acid(e.g., a C12), a fatty aldehyde (e.g., a C12), isopropanol,1,3-butanediol, crotyl alcohol, butadiene, 3-buten-2-ol, 1,4-butanediol,adipate, 6-aminocaproate, caprolactam, hexamethylenediamine, methacylicacid and 2-hydroxyisobutyric acid are thus made possible by combiningthe steps for methanol oxidation, formaldehyde fixation, formatereutilization, and product synthesis.

12 CH₄O→C₁₂H₂₆O+11 H₂O  (Fatty Alcohol on MeOH)

12 CH₄O+O₂→C₁₂H₂₄O₂+12 H₂O  (Fatty Acid on MeOH)

12 CH₄O+0.5 O₂→C₁₂H₂₄O+12 H₂O  (Fatty Aldehyde on MeOH)

4 CH₄O+1.5 O₂→C₃H₈O+4 H₂O+CO₂  (Isopropanol on MeOH)

Additional enhanced maximum yield stoichiometnes can be found in thetable below. These stoichiometnes assume that the carbon generated fromconverting pyruvate to acetyl-CoA is recycled back into product and notemitted as CO2.

Product CH4O O2 NH3 Product H2O CO2 1,3-Butanediol 4.000 0.500 0.000 -->1.000 3.000 0.000 Crotyl Alcohol 4.000 0.500 0.000 --> 1.000 4.000 0.0003-Buten-2-ol 4.000 0.500 0.000 --> 1.000 4.000 0.000 Butadiene 4.0000.500 0.000 --> 1.000 5.000 0.000 2-Hydroxyisobutyrate 4.000 1.500 0.000--> 1.000 4.000 0.000 Methacrylate (via 2-hydroxyisobutyrate) 4.0001.500 0.000 --> 1.000 5.000 0.000 3-Hydroxyisobutyrate (oxidative TCAcycle) 5.000 3.000 0.000 --> 1.000 6.000 1.000 Methacrylate (via3-hydroxyisobutyrate) 5.000 3.000 0.000 --> 1.000 7.000 1.0001,4-Butanediol (oxidative TCA cycle) 5.000 2.000 0.000 --> 1.000 5.0001.000 Adipate (oxidative TCA cycle) 7.000 4.000 0.000 --> 1.000 9.0001.000 6-Aminocaproate (oxidative TCA cycle) 7.000 3.000 1.000 --> 1.0009.000 1.000 Caprolactam (via 6-aminocaproate) 7.000 3.000 1.000 -->1.000 10.000 1.000 Hexamethylenediamine (oxidative TCA cycle) 7.0002.000 2.000 --> 1.000 9.000 1.000

By combining pathways for formaldehyde fixation and formatereutilization, yield increases on additional substrates are alsoavailable including but not limited to glucose, glycerol, sucrose,fructose, xylose, arabinose and galactose. For example, the followingmaximum theoretical yield stoichiometries for a fatty alcohol (e.g., aC12), a fatty acid (e.g., a C12), a fatty aldehyde (e.g., a C12) andisopropanol on glucose are made possible by combining the steps forformaldehyde fixation, formate reutilization, and compound synthesis.

3 C₆H₁₂O₆→C₁₂H₂₆O+5 H₂O+6 CO₂  (Fatty Alcohol on glucose)

3 C₆H₁₂O₆→1.0588 C₁₂H₂₄O₂+5.2941 H₂O+5.2941 CO₂  (Fatty Acid on glucose)

3 C₆H₁₂O₆→1.0286 C₁₂H₂₄O+5.6571 H₂O+5.6571 CO₂  (Fatty Aldehyde onglucose)

C₆H₁₂O₆→1.3333 C₃H₈O+0.6667 H₂O+2 CO₂  (Isopropanol on glucose)

Similar yield increases are observed for 1,3-butanediol, crotyl alcohol,butadiene, 3-buten-2-ol, 1,4-butanediol, adipate, 6-aminocaproate,caprolactam, hexamethylenediamine, methacylic acid, 3-hydroxyisobutricacid and 2-hydroxyisobutyric acid when formaldehyde fixation and formatereutilization pathways are used in conjunction with glycolysis, the TCAcycle, and the pentose phosphate pathway.

Product C6H12O6 O2 NH3 Product H2O CO2 1,3-Butanediol 1.000 0.000 0.000→ 1.091 0.545 1.636 Crotyl Alcohol 1.000 0.000 0.000 → 1.091 1.636 1.6363-Buten-2-ol 1.000 0.000 0.000 → 1.091 1.636 1.636 Butadiene 1.000 0.1070.000 → 1.071 2.786 1.714 2-Hydroxyisobutyrate 1.000 0.000 0.000 → 1.3330.667 0.667 Methacrylate (via 2-hydroxyisobutyrate) 1.000 0.000 0.000 →1.333 2.000 0.667 3-Hydroxyisobutyrate (oxidative TCA cycle) 1.000 0.2140.000 → 1.286 0.857 0.857 Methacrylate (via 3-hydroxyisobutyrate) 1.0000.214 0.000 → 1.286 2.143 0.857 1,4-Butanediol (oxidative TCA cycle)1.000 0.107 0.000 → 1.071 0.643 1.714 Adipate (oxidative TCA cycle)1.000 0.168 0.000 → 0.897 1.514 0.617 6-Aminocaproate (oxidative TCAcycle) 1.000 0.000 0.800 → 0.800 2.000 1.200 Caprolactam (via6-aminocaproate) 1.000 0.000 0.800 → 0.800 2.800 1.200Hexamethylenediamine (oxidative TCA cycle) 1.000 0.050 1.400 → 0.7002.500 1.800

Similarly, the maximum theoretical yield of a bioderived compound fromglycerol can be increased by enabling fixation of formaldehyde fromgeneration and utilization of formate. The following maximum theoreticalyield stoichiometries for a fatty alcohol (e.g., a C12), a fatty acid(e.g., a C12), a fatty aldehyde (e.g., a C12) and isopropanol onglycerol are thus made possible by combining the steps for formaldehydefixation, formate reutilization, and product synthesis.

6 C₃H₈O₃→1.1667 C₁₂H₂₆O+8.8333 H₂O+4 CO₂  (Fatty Alcohol on glycerol)

6 C₃H₈O₃→1.2353 C₁₂H₂₄O₂+9.1765 H₂O+3.1765 CO₂  (Fatty Acid on glycerol)

6 C₃H₈O₃→1.2000 C₁₂H₂₄O+9.6000 H₂O+3.6000 CO₂  (Fatty Aldehyde onglycerol)

C₃H₈O₃→0.7778 C₃H₈O+0.8889 H₂O+0.6667 CO₂  (Isopropanol on glycerol)

In numerous engineered pathways, product yields based on carbohydratefeedstock are hampered by insufficient reducing equivalents or by lossof reducing equivalents to byproducts. Methanol is a relativelyinexpensive organic feedstock that can be used to generate reducingequivalents by employing one or more methanol metabolic enzymes as shownin FIG. 2. Reducing equivalents can also be extracted from hydrogen andcarbon monoxide by employing hydrogenase and carbon monoxidedehydrogenase enzymes, respectively, as shown in FIG. 2. The reducingequivalents are then passed to acceptors such as oxidized ferredoxins,oxidized quinones, oxidized cytochromes, NAD(P)+, water, or hydrogenperoxide to form reduced ferredoxin, reduced quinones, reducedcytochromes, NAD(P)H, H₂, or water, respectively. Reduced ferredoxin,reduced quinones and NAD(P)H are particularly useful as they can serveas redox carriers for various Wood-Ljungdahl pathway, reductive TCAcycle, or product pathway enzymes.

The reducing equivalents produced by the metabolism of methanol,hydrogen, and carbon monoxide can be used to power several bioderivedcompound production pathways. For example, the maximum theoretical yieldof a fatty alcohol, a fatty acid, a fatty aldehyde, isopropanol,1,3-butanediol, crotyl alcohol, butadiene, 3-buten-2-ol, 1,4-butanediol,adipate, 6-aminocaproate, caprolactam, hexamethylenediamine, methacylicacid or 2-hydroxyisobutyric acid from glucose and glycerol can beincreased by enabling fixation of formaldehyde, formate reutilization,and extraction of reducing equivalents from an external source such ashydrogen. In fact, by combining pathways for formaldehyde fixation,formate reutilization, reducing equivalent extraction, and productsynthesis, the following maximum theoretical yield stoichiometries forfatty alcohol, a fatty acid, a fatty aldehyde, and isopropanol onglucose and glycerol are made possible.

2 C₆H₁₂O₆+12 H₂→C₁₂H₂₆O+11 H₂O  (Fatty Alcohol on glucose+externalredox)

2 C₆H₁₂O₆+10 H₂→C₁₂H₂₄O₂+10 H₂O  (Fatty Acid on glucose+external redox)

2 C₆H₁₂O₆+11 H₂→C₁₂H₂₄O+11 H₂O  (Fatty Aldehyde on glucose+externalredox)

C₆H₁₂O₆+6 H₂→2 C₃H₈O+4 H₂O  (Isopropanol on glucose+external redox)

4 C₃H₈O₃+8 H₂→C₁₂H₂₆O+11 H₂O  (Fatty Alcohol on glycerol+external redox)

4 C₃H₈O₃+6 H₂→C₁₂H₂₄O₂+10 H₂O  (Fatty Acid on glycerol+external redox)

4 C₃H₈O₃+7 H₂→C₁₂H₂₄O+11 H₂O  (Fatty Aldehyde on glycerol+externalredox)

C₃H₈O₃+2 H₂→C₃H₈O+2 H₂O  (Isopropanol on glycerol+external redox)

In most instances, achieving such maximum yield stoichiometries mayrequire some oxidation of reducing equivalents (e.g., H₂+½O₂→H₂O,CO+½O₂→CO₂, CH₄+1.5 O₂→CO₂+2 H₂O, C₆H₁₂O₆+6 O₂→6 CO₂+6 H₂O) to providesufficient energy for the substrate to product pathways to operate.Nevertheless, if sufficient reducing equivalents are available, enablingpathways for fixation of formaldehyde, formate reutilization, extractionof reducing equivalents, and product synthesis can even lead toproduction of a fatty alcohol, a fatty acid, a fatty aldehyde,isopropanol, and their intermediates, directly from CO₂.

Pathways identified herein, and particularly pathways exemplified inspecific combinations presented herein, are superior over other pathwaysbased in part on the applicant's ranking of pathways based on attributesincluding maximum theoretical compound yield, maximal carbon flux,maximal production of reducing equivalents, minimal production of CO₂,pathway length, number of non-native steps, thermodynamic feasibility,number of enzymes active on pathway substrates or structurally similarsubstrates, and having steps with currently characterized enzymes, andfurthermore, the latter pathways are even more favored by having inaddition at least the fewest number of non-native steps required, themost enzymes known active on pathway substrates or structurally similarsubstrates, and the fewest total number of steps from centralmetabolism.

The non-naturally occurring microbial organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

In the case of gene disruptions, a particularly useful stable geneticalteration is a gene deletion. The use of a gene deletion to introduce astable genetic alteration is particularly useful to reduce thelikelihood of a reversion to a phenotype prior to the geneticalteration. For example, stable growth-coupled production of abiochemical can be achieved, for example, by deletion of a gene encodingan enzyme catalyzing one or more reactions within a set of metabolicmodifications. The stability of growth-coupled production of abiochemical can be further enhanced through multiple deletions,significantly reducing the likelihood of multiple compensatoryreversions occurring for each disrupted activity.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of Mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having acetyl-CoA or a bioderivedcompound biosynthetic capability, those skilled in the art willunderstand with applying the teaching and guidance provided herein to aparticular species that the identification of metabolic modificationscan include identification and inclusion or inactivation of orthologs.To the extent that paralogs and/or nonorthologous gene displacements arepresent in the referenced microorganism that encode an enzyme catalyzinga similar or substantially similar metabolic reaction, those skilled inthe art also can utilize these evolutionally related genes. Similarlyfor a gene disruption, evolutionally related genes can also be disruptedor deleted in a host microbial organism to reduce or eliminatefunctional redundancy of enzymatic activities targeted for disruption.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff 50;expect: 10.0; wordsize: 3; filter on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff 50; expect: 10.0; wordsize: 11; filter off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having a methanol metabolic pathway and an acetyl-CoApathway as depicted in FIGS. 1 and 2. In some embodiments, the methanolmetabolic pathway comprises 2A or 2J, wherein 2A is a methanolmethyltransferase and 2J is a methanol dehydrogenase. In someembodiments, the methanol metabolic pathway comprises 2A. In someembodiments, the methanol metabolic pathway comprises 2J. In someembodiments, the acetyl-CoA pathway comprises a pathway selected from:(1) 1T and 1V; (2) 1T, 1W, and 1X; (3) 1U and 1V; (4) 1U, 1W, and 1X;wherein 1T is a fructose-6-phosphate phosphoketolase, wherein 1U is axylulose-5-phosphate phosphoketolase, wherein 1V is aphosphotransacetylase, wherein 1W is an acetate kinase, wherein 1X is anacetyl-CoA transferase, an acetyl-CoA synthetase, or an acetyl-CoAligase. In some embodiments, the acetyl-CoA pathway comprises (1) 1T and1V. In some embodiments, the acetyl-CoA pathway comprises (2) 1T, 1W,and 1X. In some embodiments, the acetyl-CoA pathway comprises (3) 1U and1V. In some embodiments, the acetyl-CoA pathway comprises (4) 1U, 1W,and 1X. In some embodiments, an enzyme of the methanol metabolic pathwayor the acetyl-CoA pathway is encoded by at least one exogenous nucleicacid and is expressed in a sufficient amount to enhance carbon fluxthrough acetyl-CoA.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism as described herein further comprising a formaldehydefixation pathway as depicted in FIG. 1. In some embodiments, theformaldehyde fixation pathway comprises: (1) 1D and 1Z; (2) 1D; or (3)1B and 1C, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1Cis a 6-phospho-3-hexuloisomerase, wherein 1D is a dihydroxyacetonesynthase, wherein 1Z is a fructose-6-phosphate aldolase. In someembodiments, the formaldehyde fixation pathway comprises (1) 1D and 1Z.In some embodiments, the formaldehyde fixation pathway comprises (2) 1D.In some embodiments, the formaldehyde fixation pathway comprises (3) 1Band 1C. In some embodiments, an enzyme of the formaldehyde fixationpathway is encoded by at least one exogenous nucleic acid and isexpressed in a sufficient amount to enhance carbon flux throughacetyl-CoA.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism as described herein further comprising a methanolmetabolic pathway selected from: (1) 2A and 2B; (2) 2A, 2B and 2C; (3)2J, 2K and 2C; (4) 2J, 2M, and 2N; (5) 2J and 2L; (6) 2J, 2L, and 2G;(7) 2J, 2L, and 2I; (8) 2A, 2B, 2C, 2D, and 2E; (9) 2A, 2B, 2C, 2D, and2F; (10) 2J, 2K, 2C, 2D, and 2E; (11) 2J, 2K, 2C, 2D, and 2F; (12) 2J,2M, 2N, and 2O; (13) 2A, 2B, 2C, 2D, 2E, and 2G; (14) 2A, 2B, 2C, 2D,2F, and 2G; (15) 2J, 2K, 2C, 2D, 2E, and 2G; (16) 2J, 2K, 2C, 2D, 2F,and 2G; (17) 2J, 2M, 2N, 2O, and 2G; (18) 2A, 2B, 2C, 2D, 2E, and 2I;(19) 2A, 2B, 2C, 2D, 2F, and 2I; (20) 2J, 2K, 2C, 2D, 2E, and 2I; (21)2J, 2K, 2C, 2D, 2F, and 2I; and (22) 2J, 2M, 2N, 2O, and 2I, wherein 2Ais a methanol methyltransferase, wherein 2B is amethylenetetrahydrofolate reductase, wherein 2C is amethylenetetrahydrofolate dehydrogenase, wherein 2D is amethenyltetrahydrofolate cyclohydrolase, wherein 2E is aformyltetrahydrofolate deformylase, wherein 2F is aformyltetrahydrofolate synthetase, wherein 2G is a formate hydrogenlyase, wherein 2I is a formate dehydrogenase, wherein 2J is a methanoldehydrogenase, wherein 2K is a formaldehyde activating enzyme orspontaneous, wherein 2L is a formaldehyde dehydrogenase, wherein 2M is aS-(hydroxymethyl)glutathione synthase or spontaneous, wherein 2N is aglutathione-dependent formaldehyde dehydrogenase, and wherein 2O is aS-formylglutathione hydrolase. In some embodiments, the methanolmetabolic pathway comprises (1) 2A and 2B. In some embodiments, themethanol metabolic pathway comprises (2) 2A, 2B and 2C. In someembodiments, the methanol metabolic pathway comprises (3) 2J, 2K and 2C.In some embodiments, the methanol metabolic pathway comprises (4) 2J,2M, and 2N. In some embodiments, the methanol metabolic pathwaycomprises (5) 2J and 2L. In some embodiments, the methanol metabolicpathway comprises (6) 2J, 2L, and 2G. In some embodiments, the methanolmetabolic pathway comprises (7) 2J, 2L, and 2I. In some embodiments, themethanol metabolic pathway comprises (8) 2A, 2B, 2C, 2D, and 2E. In someembodiments, the methanol metabolic pathway comprises (9) 2A, 2B, 2C,2D, and 2F. In some embodiments, the methanol metabolic pathwaycomprises (10) 2J, 2K, 2C, 2D, and 2E. In some embodiments, the methanolmetabolic pathway comprises (11) 2J, 2K, 2C, 2D, and 2F. In someembodiments, the methanol metabolic pathway comprises (12) 2J, 2M, 2N,and 2O. In some embodiments, the methanol metabolic pathway comprises(13) 2A, 2B, 2C, 2D, 2E, and 2G; (14) 2A, 2B, 2C, 2D, 2F, and 2G. Insome embodiments, the methanol metabolic pathway comprises (15) 2J, 2K,2C, 2D, 2E, and 2G. In some embodiments, the methanol metabolic pathwaycomprises (16) 2J, 2K, 2C, 2D, 2F, and 2G. In some embodiments, themethanol metabolic pathway comprises (17) 2J, 2M, 2N, 2O, and 2G. Insome embodiments, the methanol metabolic pathway comprises (18) 2A, 2B,2C, 2D, 2E, and 2I. In some embodiments, the methanol metabolic pathwaycomprises (19) 2A, 2B, 2C, 2D, 2F, and 2I. In some embodiments, themethanol metabolic pathway comprises (20) 2J, 2K, 2C, 2D, 2E, and 2I. Insome embodiments, the methanol metabolic pathway comprises (21) 2J, 2K,2C, 2D, 2F, and 2I. In some embodiments, the methanol metabolic pathwaycomprises (22) 2J, 2M, 2N, 2O, and 2I.

In some embodiments, the non-naturally occurring microbial organismdescribed herein comprises one, two, three, four, five, or six exogenousnucleic acids each encoding a methanol metabolic pathway enzyme. In someembodiments, the non-naturally occurring microbial organism describedherein comprises exogenous nucleic acids encoding each of the enzymes ofat least one of the pathways selected from (1)-(22) as describe above.In some embodiments, the non-naturally occurring microbial organismdescribed herein comprises one, two, or three exogenous nucleic acidseach encoding an acetyl-CoA pathway enzyme. In some embodiments, thenon-naturally occurring microbial organism described herein comprisesexogenous nucleic acids encoding each of the enzymes of at least one ofthe acetyl-CoA pathway selected from (1)-(4) described above.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism as described herein further comprising a formateassimilation pathway as depicted in FIG. 1. In some embodiments, theformate assimilation pathway comprises a pathway selected from: (1) 1E;(2) 1F, and 1G; (3) 1H, 1I, 1J, and 1K; (4) 1H, 1I, 1J, 1L, 1M, and 1N;(5) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (6) 1F, 1G, 1H, 1I, 1J, 1L, 1M, and1N; (7) 1K, 1H, 1I, 1J, 1L, 1M, and 1N; and (8) 1H, 1I, 1J, 1O, and 1P,wherein 1E is a formate reductase, 1F is a formate ligase, a formatetransferase, or a formate synthetase, wherein 1G is a formyl-CoAreductase, wherein 1H is a formyltetrahydrofolate synthetase, wherein 1Iis a methenyltetrahydrofolate cyclohydrolase, wherein 1J is amethylenetetrahydrofolate dehydrogenase, wherein 1K is aformaldehyde-forming enzyme or spontaneous, wherein 1L is a glycinecleavage system, wherein 1M is a serine hydroxymethyltransferase,wherein 1N is a serine deaminase, wherein 1O is amethylenetetrahydrofolate reductase, and wherein 1P is an acetyl-CoAsynthase. In some embodiments, the formate assimilation pathwaycomprises (1) 1E. In some embodiments, the formate assimilation pathwaycomprises (2) IF, and 1G. In some embodiments, the formate assimilationpathway comprises (3) 1H, 1I, 1J, and 1K. In some embodiments, theformate assimilation pathway comprises (4) 1H, 1I, 1J, 1L, 1M, and 1N.In some embodiments, the formate assimilation pathway comprises (5) 1E,1H, 1I, 1J, 1L, 1M, and 1N. In some embodiments, the formateassimilation pathway comprises (6) 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N.In some embodiments, the formate assimilation pathway comprises (7) 1K,1H, 1I, 1J, 1L, 1M, and 1N. In some embodiments, the formateassimilation pathway comprises (8) 1H, 1I, 1J, 1O, and 1P. In someembodiments, an enzyme of the formate assimilation pathway is encoded byat least one exogenous nucleic acid and is expressed in a sufficientamount to enhance carbon flux through acetyl-CoA.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having a formaldehyde fixation pathway, a formateassimilation pathway and/or an acetyl-CoA pathway as described here.Accordingly, in some embodiments, the non-naturally occurring microbialorganism of the invention can have an acetyl-CoA pathway as depicted inFIG. 3. In some embodiments, the non-naturally occurring microbialorganism of the invention can have a formaldehyde fixation pathway andan acetyl-CoA pathway as depicted in FIG. 1. In some embodiments, thenon-naturally occurring microbial organism of the invention can have aformate assimilation pathway and an acetyl-CoA pathway as depicted inFIG. 1. In some embodiments, the non-naturally occurring microbialorganism of the invention can have a formaldehyde fixation pathway, aformate assimilation pathway and an acetyl-CoA pathway as depicted inFIG. 1. In some embodiments, the formaldehyde fixation pathwaycomprises: (1) 1D and 1Z; (2) 1D; or (3) 1B and 1C, wherein 1B is a3-hexulose-6-phosphate synthase, wherein 1C is a6-phospho-3-hexuloisomerase, wherein 1D is a dihydroxyacetone synthase,wherein 1Z is a fructose-6-phosphate aldolase. In some embodiments, theformaldehyde fixation pathway comprises (1) 1D and 1Z. In someembodiments, the formaldehyde fixation pathway comprises (2) 1D. In someembodiments, the formaldehyde fixation pathway comprises (3) 1B and 1C.In some embodiment, the formate assimilation pathway comprises a pathwayselected from: (4) 1E; (5) 1F, and 1G; (6) 1H, 1I, 1J, and 1K; (7) 1H,1I, 1J, 1L, 1M, and 1N; (8) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (9) 1F, 1G,1H, 1I, 1J, 1L, 1M, and 1N; (10) 1K, 1H, 1I, 1J, 1L, 1M, and 1N; and(11) 1H, 1I, 1J, 1O, and 1P, wherein 1E is a formate reductase, 1F is aformate ligase, a formate transferase, or a formate synthetase, wherein1G is a formyl-CoA reductase, wherein 1H is a formyltetrahydrofolatesynthetase, wherein 1I is a methenyltetrahydrofolate cyclohydrolase,wherein 1J is a methylenetetrahydrofolate dehydrogenase, wherein 1K is aformaldehyde-forming enzyme or spontaneous, wherein 1L is a glycinecleavage system, wherein 1M is a serine hydroxymethyltransfemse, wherein1N is a serine deaminase, wherein 1O is a methylenetetrahydrofolatereductase, and wherein 1P is an acetyl-CoA synthase. In someembodiments, the formate assimilation pathway comprises (4) 1E. In someembodiments, the formate assimilation pathway comprises (5) 1F, and 1G.In some embodiments, the formate assimilation pathway comprises (6) 1H,1I, 1J, and 1K. In some embodiments, the formate assimilation pathwaycomprises (7) 1H, 1I, 1J, 1L, 1M, and 1N. In some embodiments, theformate assimilation pathway comprises (8) 1E, 1H, 1I, 1J, 1L, 1M, and1N. In some embodiments, the formate assimilation pathway comprises (9)1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N. In some embodiments, the formateassimilation pathway comprises (10) 1K, 1H, 1I, 1J, 1L, 1M, and 1N. Insome embodiments, the formate assimilation pathway comprises (11) 1H,1I, 1J, 1O, and 1P. In some embodiments, acetyl-CoA pathway comprises apathway selected from: (12) 1T and 1V; (13) 1T, 1W, and 1X; (14) 1U and1V; and (15) 1U, 1W, and 1X, wherein 1T is a fructose-6-phosphatephosphoketolase, wherein 1U is a xylulose-5-phosphate phosphoketolase,wherein 1V is a phosphotransacetylase, wherein 1W is an acetate kinase,wherein 1X is an acetyl-CoA transferase, an acetyl-CoA synthetase, or anacetyl-CoA ligase. In some embodiments, acetyl-CoA pathway comprises(12) 1T and 1V. In some embodiments, acetyl-CoA pathway comprises (13)1T, 1W, and 1X. In some embodiments, acetyl-CoA pathway comprises (14)1U and 1V. In some embodiments, acetyl-CoA pathway comprises (15) 1U,1W, and 1X. In some embodiments, the non-naturally occurring microbialorganism described herein comprises an acetyl-CoA pathway that comprises1T and 1V and a formaldehyde fixation pathway that comprises 1D and 1Z.In some embodiments, the non-naturally occurring microbial organismdescribed herein comprises an acetyl-CoA pathway that comprises 1T and1V and a formaldehyde fixation pathway comprises 1B and 1C. In someembodiments, an enzyme of the formaldehyde fixation pathway, the formateassimilation pathway, and/or the acetyl-CoA pathway is encoded by atleast one exogenous nucleic acid and is expressed in a sufficient amountto enhance carbon flux through acetyl-CoA.

In some embodiments, the non-naturally occurring microbial organismdescribed herein comprises one or two exogenous nucleic acids eachencoding an formaldehyde fixation pathway enzyme. In some embodiments,the non-naturally occurring microbial organism described hereincomprises one, two, three, four, five, six, seven or eight exogenousnucleic acids each encoding a formate assimilation pathway enzyme. Insome embodiments, the non-naturally occurring microbial organismdescribed herein comprises one, two, or three exogenous nucleic acidseach encoding an acetyl-CoA pathway enzyme. In some embodiments, thenon-naturally occurring microbial organism described herein comprisesexogenous nucleic acids encoding each of the enzymes of at least one ofthe pathways selected from (1)-(15) as described above.

In some embodiments, the invention further provides a non-naturallyoccurring microbial organism described herein that has a formateassimilation pathway further comprises: (1) 1Q; (2) 1R and 1S, (3) 1Yand 1Q; or (4) 1Y, 1R, and 1S, as depicted in FIG. 1, wherein 1Q is apyruvate formate lyase, wherein 1R is a pyruvate dehydrogenase, apyruvate ferredoxin oxidoreductase, or a pyruvate:NADP+ oxidoreductase,wherein 1S is a formate dehydrogenase, wherein 1Y is aglyceraldehyde-3-phosphate dehydrogenase or an enzyme of lowerglycolysis. In some embodiments, the formate assimilation pathwayfurther comprises (1) 1Q. In some embodiments, the formate assimilationpathway further comprises (2) 1R and 1S. In some embodiments, theformate assimilation pathway further comprises (3) 1Y and 1. In someembodiments, the formate assimilation pathway further comprises (4) 1Y,1R, and 1S.

In some embodiments, a non-naturally occurring microbial organism of theinvention includes a methanol oxidation pathway. Such a pathway caninclude at least one exogenous nucleic acid encoding a methanoloxidation pathway enzyme expressed in a sufficient amount to produceformaldehyde in the presence of methanol. An exemplary methanoloxidation pathway enzyme is a methanol dehydrogenase. Accordingly, insome embodiments, a non-naturally occurring microbial organism of theinvention includes at least one exogenous nucleic acid encoding amethanol dehydrogenase expressed in a sufficient amount to produceformaldehyde in the presence of methanol.

In some embodiments, the exogenous nucleic acid encoding an methanoldehydrogenase is expressed in a sufficient amount to produce an amountof formaldehyde greater than or equal to 1 μM, 10 μM, 20 μM, or 50 μM,or a range thereof, in culture medium or intracellularly. In otherembodiments, the exogenous nucleic acid encoding an methanoldehydrogenase is capable of producing an amount of formaldehyde greaterthan or equal to 1 μM, 10 μM, 20 μM, or 50 μM, or a range thereof, inculture medium or intracellularly. In some embodiments, the range isfrom 1 μM to 50 μM or greater. In other embodiments, the range is from10 μM to 50 μM or greater. In other embodiments, the range is from 20 μMto 50 μM or greater. In other embodiments, the amount of formaldehydeproduction is 50 μM or greater. In specific embodiments, the amount offormaldehyde production is in excess of, or as compared to, that of anegative control, e.g., the same species of organism that does notcomprise the exogenous nucleic acid, such as a wild-type microbialorganism or a control microbial organism thereof. In certainembodiments, the methanol dehydrogenase is selected from those providedherein, e.g., as exemplified in Example II (see FIG. 1, Step A, or FIG.10, Step J). In certain embodiments, the amount of formaldehydeproduction is determined by a whole cell assay, such as that provided inExample II (see FIG. 1, Step A, or FIG. 10, Step J), or by another assayprovided herein or otherwise known in the art. In certain embodiments,formaldehyde utilization activity is absent in the whole cell.

In certain embodiments, the exogenous nucleic acid encoding an methanoldehydrogenase is expressed in a sufficient amount to produce at least1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 30×, 40×, 50×, 100×or more formaldehyde in culture medium or intracellularly. In otherembodiments, the exogenous nucleic acid encoding an methanoldehydrogenase is capable of producing an amount of formaldehyde at least1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 30×, 40×, 50×, 100×,or a range thereof, in culture medium or intracellularly. In someembodiments, the range is from 1× to 100×. In other embodiments, therange is from 2× to 100×. In other embodiments, the range is from 5× to100×. In other embodiments, the range is from 10× to 100×. In otherembodiments, the range is from 50× to 100×. In some embodiments, theamount of formaldehyde production is at least 20×. In other embodiments,the amount of formaldehyde production is at least 50×. In specificembodiments, the amount of formaldehyde production is in excess of, oras compared to, that of a negative control, e.g, the same species oforganism that does not comprise the exogenous nucleic acid, such as awild-type microbial organism or a control microbial organism thereof. Incertain embodiments, the methanol dehydrogenase is selected from thoseprovided herein, e.g, as exemplified herein (see FIG. 1, Step A, or FIG.2, Step J). In certain embodiments, the amount of formaldehydeproduction is determined by a whole cell assay, such as that providedherein (see FIG. 1, Step A, or FIG. 2, Step J), or by another assayprovided herein or otherwise known in the art. In certain embodiments,formaldehyde utilization activity is absent in the whole cell.

In some embodiments, a non-naturally occurring microbial organism of theinvention includes one or more enzymes for generating reducingequivalents. For example, the microbial organism can further include ahydrogenase and/or a carbon monoxide dehydrogenase. In some aspects, theorganism comprises an exogenous nucleic acid encoding the hydrogenase orthe carbon monoxide dehydrogenase.

A reducing equivalent can also be readily obtained from a glycolysisintermediate by any of several central metabolic reactions includingglyceraldehyde-3-phosphate dehydrogenase, pyruvate dehydrogenase,pyruvate formate lyase and NAD(P)-dependent formate dehydrogenase,isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinatedehydrogenase, and malate dehydrogenase. Additionally, reducingequivalents can be generated from glucose 6-phosphate-1-dehydrogenaseand 6-phosphogluconate dehydrogenase of the pentose phosphate pathway.Overall, at most twelve reducing equivalents can be obtained from a C6glycolysis intermediate (e.g., glucose-6-phosphate,fructose-6-phosphate, fructose-1,6-diphosphate) and at most six reducingequivalents can be generated from a C3 glycolysis intermediate (e.g.,dihydroxyacetone phosphate, glyceraldehyde-3-phosphate).

In some embodiments, the at least one exogenous nucleic acid included inthe non-naturally occurring microbial organism of the invention is aheterologous nucleic acid. Accordingly, in some embodiments, the atleast one exogenous nucleic acid encoding a formaldehyde fixationpathway enzyme described herein is a heterologous nucleic acid. In someembodiments, the at least one exogenous nucleic acid encoding a formateassimilation pathway enzyme described herein is a heterologous nucleicacid. In some embodiments, the at least one exogenous nucleic acidencoding a methanol metabolic pathway enzyme described herein is aheterologous nucleic acid. In some embodiments, the at least oneexogenous nucleic acid encoding a methanol oxidation pathway enzymedescribed herein is a heterologous nucleic acid. In some embodiments,the at least one exogenous nucleic acid encoding a hydrogenase or acarbon monoxide dehydrogenase is a heterologous nucleic acid.

In some embodiments, the non-naturally occurring microbial organism ofthe invention is in a substantially anaerobic culture medium.

In some embodiments, a non-naturally occurring microbial organismdescribed herein further includes a pathway capable of producingsuccinyl-CoA, malonyl-CoA, and/or acetoacetyl-CoA, wherein the pathwayconverts acetyl-CoA to succinyl-CoA, malonyl-CoA, and/or acetoacetyl-CoAby one or more enzymes. Accordingly, in some embodiments the microbialorganism includes a succinyl-CoA pathway, wherein the pathway convertsacetyl-CoA to the succinyl-CoA by one or more enzymes. In someembodiments, the microbial organism includes a malonyl-CoA pathway,wherein the pathway converts acetyl-CoA to malonyl-CoA by one or moreenzymes. In some embodiments, the microbial organism includes anacetoacetyl-CoA pathway, wherein the pathway converts acetyl-CoA toacetoacetyl-CoA by one or more enzymes.

In some embodiments, the invention provides that a non-naturallyoccurring microbial organism as described herein further includes apathway capable of producing a bioderived compound as described herein.In some aspects, the bioderived compound is an alcohol, a glycol, anorganic acid, an alkene, a diene, an organic amine, an organic aldehyde,a vitamin, a nutraceutical or a pharmaceutical.

In some embodiments, the non-naturally occurring microbial organism ofthe invention includes a pathway for production of an alcohol asdescribed herein. Accordingly, in some embodiments, the alcohol isselected from: (i) a biofuel alcohol, wherein said biofuel is a primaryalcohol, a secondary alcohol, a diol or triol comprising C3 to C10carbon atoms; (ii) n-propanol or isopropanol; and (iii) a fatty alcohol,wherein said fatty alcohol comprises C4 to C27 carbon atoms, C8 to C18carbon atoms, C12 to C18 carbon atoms, or C12 to C14 carbon atoms. Insome aspects, the biofuel alcohol is selected from 1-propanol,isopropanol, 1-butanol, isobutanol, 1-pentanol, isopentenol,2-methyl-1-butanol, 3-methyl-1-butanol, 1-hexanol, 3-methyl-1-pentanol,1-heptanol, 4-methyl-1-hexanol, and 5-methyl-1-hexanol.

In some embodiments, the non-naturally occurring microbial organism ofthe invention includes a pathway for production of an diol. Accordingly,in some embodiments, the diol is a propanediol or a butanediol. In someaspects, the butanediol is 1,4 butanediol, 1,3-butanediol or2,3-butanediol.

In some embodiments, the non-naturally occurring microbial organism ofthe invention includes a pathway for production of a bioderived compoundselected from: (i) 1,4-butanediol or an intermediate thereto, whereinsaid intermediate is optionally 4-hydroxybutanoic acid (4-HB); (ii)butadiene (1,3-butadiene) or an intermediate thereto, wherein saidintermediate is optionally 1,4-butanediol, 1,3-butanediol,2,3-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) or3-buten-1-ol; 1,3-butanediol or an intermediate thereto, wherein saidintermediate is optionally 3-hydroxybutyrate (3-HB), 2,4-pentadienoate,crotyl alcohol or 3-buten-1-ol; (iv) adipate, 6-aminocaproic acid,caprolactam, hexamethylenediamine, levulinic acid or an intermediatethereto, wherein said intermediate is optionally adipyl-CoA or4-aminobutyryl-CoA; (v) methacrylic acid or an ester thereof,3-hydroxyisobutyrate, 2-hydroxyisobutyrate, or an intermediate thereto,wherein said ester is optionally methyl methacrylate or poly(methylmethacrylate); (vi) 1,2-propanediol (propylene glycol), 1,3-propanediol,glycerol, ethylene glycol, diethylene triethylene glycol, dipropyleneglycol, tripropylene glycol, neopentyl glycol, bisphenol A or anintermediate thereto; (vii) succinic acid or an intermediate thereto;and (viii) a fatty alcohol, a fatty aldehyde or a fatty acid comprisingC4 to C27 carbon atoms, C8 to C18 carbon atoms, C12 to C18 carbon atoms,or C12 to C14 carbon atoms, wherein said fatty alcohol is optionallydodecanol (C12; lauryl alcohol), tridecyl alcohol (C13; 1-tridecanol,tridecanol, isotridecanol), myristyl alcohol (C14; 1-tetradecanol),pentadecyl alcohol (C15; 1-pentadecanol, pentadecanol), cetyl alcohol(C16; 1-hexadecanol), heptadecyl alcohol (C17; 1-n-heptadecanol,heptadecanol) and stearyl alcohol (C18; 1-octadecanol) or palmitoleylalcohol (C16 unsaturated; cis-9-hexadecen-1-ol). Accordingly, in someembodiments, the non-naturally occurring microbial organism of theinvention includes a pathway for production of 1,4-butanediol or anintermediate thereto, wherein said intermediate is optionally4-hydroxybutanoic acid (4-HB). In some embodiments, the non-naturallyoccurring microbial organism of the invention includes a pathway forproduction of butadiene (1,3-butadiene) or an intermediate thereto,wherein said intermediate is optionally 1,4-butanediol, 1,3-butanediol,2,3-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) or3-buten-1-ol. In some embodiments, the non-naturally occurring microbialorganism of the invention includes a pathway for production of1,3-butanediol or an intermediate thereto, wherein said intermediate isoptionally 3-hydroxybutyrate (3-HB), 2,4-pentadienoate, crotyl alcoholor 3-buten-1-ol. In some embodiments, the non-naturally occurringmicrobial organism of the invention includes a pathway for production ofadipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine,levulinic acid or an intermediate thereto, wherein said intermediate isoptionally adipyl-CoA or 4-aminobutyryl-CoA. In some embodiments, thenon-naturally occurring microbial organism of the invention includes apathway for production of methacrylic acid or an ester thereof,3-hydroxyisobutyrate, 2-hydroxyisobutyrate, or an intermediate thereto,wherein said ester is optionally methyl methacrylate or poly(methylmethacrylate). In some embodiments, the non-naturally occurringmicrobial organism of the invention includes a pathway for production of1,2-propanediol (propylene glycol), 1,3-propanediol, glycerol, ethyleneglycol, diethylene glycol, triethylene glycol, dipropylene glycol,tripropylene glycol, neopentyl bisphenol A or an intermediate thereto.In some embodiments, the non-naturally occurring microbial organism ofthe invention includes a pathway for production of succinic acid or anintermediate thereto. In some embodiments, the non-naturally occurringmicrobial organism of the invention includes a pathway for production ofa fatty alcohol, a fatty aldehyde or a fatty acid comprising C4 to C27carbon atoms, C8 to C18 carbon atoms, C12 to C18 carbon atoms, or C12 toC14 carbon atoms, wherein said fatty alcohol is optionally dodecanol(C12; lauryl alcohol), tridecyl alcohol (C13; 1-tridecanol, tridecanol,isotridecanol), myristyl alcohol (C14; 1-tetradecanol), pentadecylalcohol (C15; 1-pentadecanol, pentadecanol), cetyl alcohol (C16;1-hexadecanol), heptadecyl alcohol (C17; 1-n-heptadecanol, heptadecanol)and stearyl alcohol (C18; 1-octadecanol) or palmitoleyl alcohol (C16unsaturated; cis-9-hexadecen-1-ol).

In some embodiments, a non-naturally occurring microbial organism of theinvention further comprises a 1,3-butanediol pathway and an exogenousnucleic acid encoding a 1,3-butanediol pathway enzyme expressed in asufficient amount to produce 1,3-butanediol as depicted in FIG. 5.Accordingly, in some embodiments, the 1,3-butanediol pathway comprises apathway selected from: (1) 5A, 5B, 5D, 5E, and 5H; (2) 5A, 5B, 5D, 5F,5G, and 5H; (3) 5C, 5D, 5E, and 5H; (4) 5C, 5D, 5F, 5G, and 5H; (5) 5A,5B, 5D and 5V; and (6) 5C, 5D and 5V wherein 5A is an acetyl-CoAcarboxylase, wherein 5B is an acetoacetyl-CoA synthase, wherein 5C is anacetyl-CoA:acetyl-CoA acyltransferase, wherein 5D is an acetoacetyl-CoAreductase (ketone reducing), wherein 5E is a 3-hydroxybutyryl-CoAreductase (aldehyde forming), wherein 5F is a 3-hydroxybutyryl-CoAhydrolase, transferase or synthetase, wherein 5G is a 3-hydroxybutyratereductase, wherein 5H is a 3-hydroxybutyraldehyde reductase, wherein 5Vis a 3-hydroxybutyryl-CoA reductase (alcohol forming).

In some embodiments, a non-naturally occurring microbial organism of theinvention further comprises a crotyl alcohol pathway and an exogenousnucleic acid encoding a crotyl alcohol pathway enzyme expressed in asufficient amount to produce crotyl alcohol as depicted in FIG. 5.Accordingly, in some embodiments, the crotyl alcohol pathway comprises apathway selected from: (1) 5A, 5B, 5D, 5J, 5K, and 5N; (2) 5A, 5B, 5D,5J, 5L, 5M, and 5N; (3) 5C, 5D, 5J, 5K, and 5N; (4) 5C, 5D, 5J, 5L, 5M,and 5N; (5) 5A, 5B, 5D, 5J and 5U; and (6) 5C, 5D, 5J and 5U, wherein 5Ais an acetyl-CoA carboxylase, wherein 5B is an acetoacetyl-CoA synthase,wherein 5C is an acetyl-CoA:acetyl-CoA acyltransferase, wherein 5D is anacetoacetyl-CoA reductase (ketone reducing), wherein 5J is a3-hydroxybutyryl-CoA dehydratase, wherein 5K is a crotonyl-CoA reductase(aldehyde forming), wherein 5L is a crotonyl-CoA hydrolase, crotonyl-CoAtransferase or crotonyl-CoA synthetase, wherein 5M is a crotonatereductase, wherein 5N is a crotonaldehyde reductase, wherein 5U is acrotonyl-CoA reductase (alcohol forming).

In some embodiments, a non-naturally occurring microbial organism of theinvention further comprises a butadiene pathway and an exogenous nucleicacid encoding a butadiene pathway enzyme expressed in a sufficientamount to produce butadiene as depicted in FIGS. 5 and 6. Accordingly,in some embodiments, the butadiene pathway comprises a pathway selectedfrom: (1) 5A, 5B, 5D, 5E, 5H, 6A, 6B, 6C, and 6G; (2) 5A, 5B, 5D, 5F,5G, 5H, 6A, 6B, 6C, and 6G; (3) 5C, 5D, 5E, 5H, 6A, 6B, 6C, and 6G; (4)5C, 5D, 5F, 5G, 5H, 6A, 6B, 6C, and 6G; (5) 5A, 5B, 5D, 5E, 5H, 6A, 6F,and 6G; (6) 5A, 5B, 5D, 5F, 5G, 5H, 6A, 6F, and 6G; (7) 5C, 5D, 5E, 5H,6A, 6F, and 6G; (8) 5C, 5D, 5F, 5G, 5H, 6A, 6F, and 6G; (9) 5A, 5B, 5D,5E, 5H, 6E, 6C, and 6G; (10) 5A, 5B, 5D, 5F, 5G, 5H, 6E, 6C, and 6G;(11) 5C, 5D, 5E, 5H, 6E, 6C, and 6G; (12) 5C, 5D, 5F, 5G, 5H, 6E, 6C,and 6G; (13) 5A, 5B, 5D, 5E, 5H, 6D, and 6G; (14) 5A, 5B, 5D, 5F, 5G,5H, 6D, and 6G; (15) 5C, 5D, 5E, 5H, 6D, and 6G; (16) 5C, 5D, 5F, 5G,5H, 6D, and 6G; (17) 5A, 5B, 5D, 5J, 5K, 5N, and 5S; (18) 5A, 5B, 5D,5J, 5L, 5M, 5N, and 5S; (19) 5C, 5D, 5J, 5K, 5N, and 5S; (20) 5C, 5D,5J, 5L, 5M, 5N, and 5S; (21) 5A, 5B, 5D, 5J, 5K, 5N, 5R, and 5Q; (22)5A, 5B, 5D, 5J, 5L, 5M, 5N, 5R, and 5Q; (23) 5C, 5D, 5J, 5K, 5N, 5R, and5Q; (24) 5C, 5D, 5J, 5L, 5M, 5N, 5R, and 5Q; (25) 5A, 5B, 5D, 5J, 5K,5N, 5O, 5P, and 5Q; (26) 5A, 5B, 5D, 5J, 5L, 5M, 5N, 5O, 5P, and 5Q;(27) 5C, 5D, 5J, 5K, 5N, 5O, 5P, and 5Q; (28) 5C, 5D, 5J, 5L, 5M, 5N,5O, 5P, and 5Q; (29) 5A, 5B, 5D, 5J, 5K, 5N, 5O, and 5T; (30) 5A, 5B,5D, 5J, 5L, 5M, 5N, 5O, and 5T; (31) 5C, 5D, 5J, 5K, 5N, 5O, and 5T;(32) 5C, 5D, 5J, 5L, 5M, 5N, 5O, and 5T, (33) 5A, 5B, 5D, 5V, 6A, 6B,6C, and 6G; (34) 5C, 5D, 5V, 6A, 6B, 6C, and 6G; (35) 5A, 5B, 5D, 5J,5U, and 5S; (36) 5C, 5D, 5J, 5U, and 5S; (37) 5A, 5B, 5D, 5J, 5U, 5R,and 5Q; (38) 5C, 5D, 5J, 5U, 5R, and 5Q; (39) 5A, 5B, 5D, 5J, 5U, 5O,5P, and 5Q; (40) 5C, 5D, 5J, 5U, 5O, 5P, and 5Q; (41) 5A, 5B, 5D, 5J,5U, 5O, and 5T; and (42) 5C, 5D, 5J, 5U, 5O, and 5T, wherein 5A is anacetyl-CoA carboxylase, wherein 5B is an acetoacetyl-CoA synthase,wherein 5C is an acetyl-CoA:acetyl-CoA acyltransferase, wherein 5D is anacetoacetyl-CoA reductase (ketone reducing), wherein 5E is a3-hydroxybutyryl-CoA reductase (aldehyde forming), wherein 5F is a3-hydroxybutyryl-CoA hydrolase, 3-hydroxybutyryl-CoA transferase or3-hydroxybutyryl-CoA synthetase, wherein 5G is a 3-hydroxybutyratereductase, wherein 5H is a 3-hydroxybutyraldehyde reductase, wherein 5Jis a 3-hydroxybutyryl-CoA dehydratase, wherein 5K is a crotonyl-CoAreductase (aldehyde forming), wherein 5L is a crotonyl-CoA hydrolase,crotonyl-CoA transferase or crotonyl-CoA synthetase, wherein 5M is acrotonate reductase, wherein 5N is a crotonaldehyde reductase, wherein50 is a crotyl alcohol kinase, wherein 5P is a 2-butenyl-4-phosphatekinase, wherein 5Q is a butadiene synthase, wherein 5R is a crotylalcohol diphosphokinase, wherein 5S is chemical dehydration or a crotylalcohol dehydratase, wherein 5T is a butadiene synthase (monophosphate),wherein 5T is a butadiene synthase (monophosphate), wherein 5U is acrotonyl-CoA reductase (alcohol forming), wherein 5V is a3-hydroxybutyryl-CoA reductase (alcohol forming), wherein 6A is a1,3-butanediol kinase, wherein 6B is a 3-hydroxybutyrylphosphate kinase,wherein 6C is a 3-hydroxybutyryldiphosphate lyase, wherein 6D is a1,3-butanediol diphosphokinase, wherein 6E is a 1,3-butanedioldehydratase, wherein 6F is a 3-hydroxybutyrylphosphate lyase, wherein 6Gis a 3-buten-2-ol dehydratase or chemical dehydration.

In some embodiments, a non-naturally occurring microbial organism of theinvention further comprises a 3-buten-2-ol pathway and an exogenousnucleic acid encoding a 3-buten-2-ol pathway enzyme expressed in asufficient amount to produce 3-buten-2-ol as depicted in FIGS. 5 and 6.Accordingly, in some embodiments, the 3-buten-2-ol pathway comprises apathway selected from: (1) 5A, 5B, 5D, 5E, 5H, 6A, 6B, and 6C; (2) 5A,5B, 5D, 5F, 5G, 5H, 6A, 6B, and 6C; (3) 5C, 5D, 5E, 5H, 6A, 6B, and 6C;(4) 5C, 5D, 5F, 5G, 5H, 6A, 6B, and 6C; (5) 5A, 5B, 5D, 5E, 5H, 6A, and6F; (6) 5A, 5B, 5D, 5F, 5G, 5H, 6A, and 6F; (7) 5C, 5D, 5E, 5H, 6A, and6F; (8) 5C, 5D, 5F, 5G, 5H, 6A, and 6F; (9) 5A, 5B, 5D, 5E, 5H, 6E, and6C; (10) 5A, 5B, 5D, 5F, 5G, 5H, 6E, and 6C; (11) 5C, 5D, 5E, 5H, 6E,and 6C; (12) 5C, 5D, 5F, 5G, 5H, 6E, and 6C; (13) 5A, 5B, 5D, 5E, 5H,and 6D; (14) 5A, 5B, 5D, 5F, 5G, 5H, and 6D; (15) 5C, 5D, 5E, 5H, and6D; (16) 5C, 5D, 5F, 5G, 5H, and 6D; (17) 5A, 5B, 5D, 5V, 6A, 6B, and6C; (18) 5C, 5D, 5V, 6A, 6B, and 6C; (19) 5A, 5B, 5D, 5V, 6A, and 6F;(20) 5C, 5D, 5V, 6A, and 6F; (21) 5A, 5B, 5D, 5V, 6E, and 6C; (22) 5C,5D, 5V, 6E, and 6C; (23) 5A, 5B, 5D, 5V and 6D; and (24) 5C, 5D, 5V and6D, wherein 5A is an acetyl-CoA carboxylase, wherein 5B is anacetoacetyl-CoA synthase, wherein 5C is an acetyl-CoA:acetyl-CoAacyltransferase, wherein 5D is an acetoacetyl-CoA reductase (ketonereducing), wherein 5E is a 3-hydroxybutyryl-CoA reductase (aldehydeforming), wherein 5F is a 3-hydroxybutyryl-CoA hydrolase,3-hydroxybutyryl-CoA transferase or 3-hydroxybutyryl-CoA synthetase,wherein 5G is a 3-hydroxybutyrate reductase, wherein 5H is a3-hydroxybutyraldehyde reductase, wherein 5V is a 3-hydroxybutyryl-CoAreductase (alcohol forming), wherein 6A is a 1,3-butanediol kinase,wherein 6B is a 3-hydroxybutyrylphosphate kinase, wherein 6C is a3-hydroxybutyryldiphosphate lyase, wherein 6D is a 1,3-butanedioldiphosphokinase, wherein 6E is a 1,3-butanediol dehydratase, wherein 6Fis a 3-hydroxybutyrylphosphate lyase.

In some embodiments, a non-naturally occurring microbial organism of theinvention further comprises a 1,4-butanediol pathway and an exogenousnucleic acid encoding a 1,4-butanediol pathway enzyme expressed in asufficient amount to produce 1,4-butanediol as depicted in FIG. 7.Accordingly, in some embodiments, the 1,4-butanediol pathway comprises apathway selected from: (1) 7B, 7C, 7D, 7E, 7F, and 7G; (2) 7A, 7H, 7C,7D, 7E, 7F, and 7G; (3) 7I, 7D, 7E, 7F, and 7G; (4) 7B, 7C, 7K, and 7G;(5) 7A, 7H, 7C, 7K, and 7G; (6) 7I, 7K, and 7G; (7) 7B, 7C, 7D, 7L, and7G; (8) 7A, 7H, 7C, 7D, 7L, and 7G; (9) 7I, 7D, 7L, and 7G; (10) 7B, 7C,7J, 7F, and 7G; (11) 7A, 7H, 7C, 7J, 7F, and 7G; (12) 7I, 7J, 7F, and7G; (13) 7B, 7C, 7D, 7E, and 7M; (14) 7A, 7H, 7C, 7D, 7E, and 7M; and(15) 7I, 7D, 7E, and 7M, wherein 7A is a succinyl-CoA transferase or asuccinyl-CoA synthetase, wherein 7B is a succinyl-CoA reductase(aldehyde forming), wherein 7C is a 4-HB dehydrogenase, wherein 7D is a4-HB kinase, wherein 7E is a phosphotrans-4-hydroxybutyrylase, wherein7F is a 4-hydroxybutyryl-CoA reductase (aldehyde forming), wherein 7G isa 1,4-butanediol dehydrogenase, wherein 7H is a succinate reductase,wherein 71 is a succinyl-CoA reductase (alcohol forming), wherein 7J isa 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA synthetase,wherein 7K is a 4-HB reductase, wherein 7L is a4-hydroxybutyryl-phosphate reductase, wherein 7M is a4-hydroxybutyryl-CoA reductase (alcohol forming).

In some embodiments, a non-naturally occurring microbial organism of theinvention further comprises an adipate pathway and an exogenous nucleicacid encoding an adipate pathway enzyme expressed in a sufficient amountto produce adipate as depicted in FIG. 8. Accordingly, in someembodiments, the adipate pathway comprises 8A, 8B, 8C, 8D and 8L,wherein 8A is a 3-oxoadipyl-CoA thiolase, wherein 8B is a3-oxoadipyl-CoA reductase, wherein 8C is a 3-hydroxyadipyl-CoAdehydratase, wherein 8D is a 5-carboxy-2-pentenoyl-CoA reductase,wherein 8L is an adipyl-CoA hydrolase, adipyl-CoA ligase, adipyl-CoAtransferase, or phosphotransadipylase/adipate kinase.

In some embodiments, a non-naturally occurring microbial organism of theinvention further comprises a 6-aminocaproate pathway and an exogenousnucleic acid encoding a 6-aminocaproate pathway enzyme expressed in asufficient amount to produce 6-aminocaproate as depicted in FIG. 8.Accordingly, in some embodiments, the 6-aminocaproate pathway comprises8A, 8B, 8C, 8D, 8E, and 8F, wherein 8A is a 3-oxoadipyl-CoA thiolase,wherein 8B is a 3-oxoadipyl-CoA reductase, wherein 8C is a3-hydroxyadipyl-CoA dehydratase, wherein 8D is a5-carboxy-2-pentenoyl-CoA reductase, wherein 8E is an adipyl-CoAreductase (aldehyde forming), wherein 8F is a 6-aminocaproatetransaminase or 6-aminocaproate dehydrogenase.

In some embodiments, a non-naturally occurring microbial organism of theinvention further includes a caprolactam pathway and an exogenousnucleic acid encoding a caprolactam pathway enzyme expressed in asufficient amount to produce caprolactam as depicted in FIG. 8.Accordingly, in some embodiments, the caprolactam pathway comprises: (1)8A, 8B, 8C, 8D, 8E, 8F, and 8H; or (2) 8A, 8B, 8C, 8D, 8E, 8F, 8G, and8I, wherein 8A is a 3-oxoadipyl-CoA thiolase, wherein 8B is a3-oxoadipyl-CoA reductase, wherein 8C is a 3-hydroxyadipyl-CoAdehydratase, wherein 8D is a 5-carboxy-2-pentenoyl-CoA reductase,wherein 8E is an adipyl-CoA reductase (aldehyde forming), wherein 8F isa 6-aminocaproate transaminase or 6-aminocaproate dehydrogenase, wherein8G is a 6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoAsynthase, wherein 8H is an amidohydrolase, wherein 81 is spontaneouscyclization.

In some embodiments, a non-naturally occurring microbial organism of theinvention further comprises a hexamethylenediamine pathway and anexogenous nucleic acid encoding a hexamethylenediamine pathway enzymeexpressed in a sufficient amount to produce hexamethylenediamine asdepicted in FIG. 8. Accordingly, in some embodiments, thehexamethylenediamine pathway comprises 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8J,8K, wherein 8A is a 3-oxoadipyl-CoA thiolase, wherein 8B is a3-oxoadipyl-CoA reductase, wherein 8C is a 3-hydroxyadipyl-CoAdehydratase, wherein 8D is a 5-carboxy-2-pentenoyl-CoA reductase,wherein 8E is an adipyl-CoA reductase (aldehyde forming), wherein 8F isa 6-aminocaproate transaminase or 6-aminocaproate dehydrogenase, wherein8G is a 6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoAsynthase, wherein 8J is a 6-aminocaproyl-CoA reductase (aldehydeforming), wherein 8K is a hexamethylenediamine transaminase orhexamethylenediamine dehydrogenase.

In some embodiments, a non-naturally occurring microbial organism of theinvention further comprises a methacrylic acid pathway and an exogenousnucleic acid encoding a methacrylic acid pathway enzyme expressed in asufficient amount to produce methacrylic acid as depicted in FIGS. 9 and10. Accordingly, in some embodiments, the methacrylic acid pathwaycomprises a pathway selected from: (1) 9A, 9B, 9C, 9D, and 9E; (2) 9A,9F, and 9E; (3) 9A, 9B, 9F, and 9E; (4) 9A, 9C, 9D, and 9E; and (5) 10A,10B, 10C, 10D, and 10E, wherein 9A is a methylmalonyl-CoA mutase,wherein 9B is a methylmalonyl-CoA epimerase, wherein 9C is amethylmalonyl-CoA reductase (aldehyde forming), wherein 9D is amethylmalonate semialdehyde reductase, wherein 9E is a3-hydroxyisobutyrate dehydratase, wherein 9F is a methylmalonyl-CoAreductase (alcohol forming), wherein 10A is an acetyl-CoA:acetyl-CoAacyltransferase, wherein 10B is an acetoacetyl-CoA reductase (ketonereducing), wherein 10C is a 3-hydroxybutyrl-CoA mutase, wherein 10D is a2-hydroxyisobutyryl-CoA dehydratase, wherein 10E is a methacrylyl-CoAsynthetase, methacrylyl-CoA hydrolase, or methacrylyl-CoA transferase.

In some embodiments, a non-naturally occurring microbial organism of theinvention further comprises a 2-hydroxyisobutyric acid pathway and anexogenous nucleic acid encoding a 2-hydroxyisobutyric acid pathwayenzyme expressed in a sufficient amount to produce 2-hydroxyisobutyricacid as depicted in FIG. 10. Accordingly, in some embodiments, the2-hydroxyisobutyric acid pathway comprises 10A, 10B, 10C, and 10F,wherein 10A is an acetyl-CoA:acetyl-CoA acyltransferase, wherein 10B isan acetoacetyl-CoA reductase (ketone reducing), wherein 10C is a3-hydroxybutyrl-CoA mutase, wherein 10F is a 2-hydroxyisobutyryl-CoAsynthetase, 2-hydroxyisobutyryl-CoA hydrolase, or2-hydroxyisobutyryl-CoA transferase.

In some embodiments, a non-naturally occurring microbial organism of theinvention further comprises a succinyl-CoA pathway and an exogenousnucleic acid encoding a succinyl-CoA pathway enzyme expressed in asufficient amount to produce succinyl-CoA as depicted in FIG. 4.Accordingly, in some embodiments, the succinyl-CoA pathway comprises apathway selected from: (1) 4H, 4I, 4J, and 4K; (2) 4H, 4I, 4N, and 4E;(3) 4H, 4I, 4N, 4C, 4D, and 4E; (4) 4L, 4H, 4I, 4J, and 4K; (5) 4L, 4H,4I, 4N, and 4E; (6) 4L, 4H, 4I, 4N, 4C, 4D, and 4E; (7) 4A, 4H, 4I, 4J,and 4K; (8) 4A, 4H, 4I, 4N, and 4E; (9) 4A, 4H, 4I, 4N, 4C, 4D, and 4E;(10) 4M, 4C, 4D, and 4E; (11) 4F, 4L, 4H, 4I, 4J, and 4K; (12) 4F, 4L,4H, 4I, 4N, and 4E; (13) 4F, 4L, 4H, 4I, 4N, 4C, 4D, and 4E; (14) 4F,4M, 4C, 4D, and 4E; (15) 4F, 4G, 4H, 4I, 4J, and 4K; (16) 4F, 4G, 4H,4I, 4N, and 4E; and (17) 4F, 4G, 4H, 4I, 4N, 4C, 4D, and 4E, wherein 4Ais a PEP carboxylase or PEP carboxykinase, wherein 4B is a malatedehydrogenase, wherein 4C is a fumarase, wherein 4D is a fumaratereductase, wherein 4E is a succinyl-CoA synthetase or succinyl-CoAtransferase, wherein 4F is a pyruvate kinase or PTS-dependent substrateimport, wherein 4G is a pyruvate dehydrogenase, pyruvate formate lyase,or pyruvate:ferredoxin oxidoreductase, wherein 4H is a citrate synthase,wherein 41 is an aconitase, wherein 4J is an isocitrate dehydrogenase,wherein 4K is an alpha-ketoglutarate dehydrogenase, wherein 4L is apyruvate carboxylase, wherein 4M is a malic enzyme, wherein 4N is anisocitrate lyase and malate synthase.

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having an acetyl-CoA or bioderived compoundpathway, wherein the non-naturally occurring microbial organismcomprises at least one exogenous nucleic acid encoding an enzyme orprotein that converts a substrate to a product selected from the groupconsisting of MeOH to Fald, Fald to H6P, H6P to F6P, Fald to DHA andG3P, DHA and G3P to F6P, F6P to ACTP and E4P, ACTP to ACCOA, ACTP toacetate, acetate to ACCOA, Xu5P to ACTP and G3P, G3P to PYR, PYR toformate and ACCOA, PYR to CO₂ and ACCOA, CO₂ to formate, formate toFald, formate to Formyl-CoA, Formyl-CoA to Fald, Formate to FTHF, FTHFto methenyl-THF, methenyl-THF to methylene-THF, methylene-THF to Fald,methylene-THF to glycine, glycine to serine, serine to PYR,methylene-THF to methyl-THF, methyl-THF to ACCOA, G3P to PEP, PEP toPYR, PYR to ACCOA, PEP to OAA, OAA to MAL, MAL to FUM, FUM to SUCC, SUCCto SUCCOA, ACCOA and OAA to CIT, CIT to ICIT, ICIT to AKG, AKG toSUCCOA, PYR to OAA, PYR to MAL, ICIT to MAL and SUCC, ACCOA to MALCOA,MALCOA and ACCOA to AACOA, ACCOA to AACOA, ACCOA to 3HBCOA, 3HBCOA to3HBALD, 3HBALD to 13BDO; 13BDO to Butadiene, 3HBCOA to CROTCOA, CROTCOAto CROTALD, CROTCOA to CROT, CROT to CROTALD, CROTALD to CROTALC,CROTALD to CROT-Pi, CROT-Pi to CROT-PPi, CROT-Ppi to butadiene, CROTALDto CROT-PPi, CROT-Pi to butadiene, 1,3-butanediol to 3-hydroxybutyrylphosphate, 3-hydroxybutyryl phosphate to 3-hydroxybutyryl diphosphate;3-hydroxybutyryl diphosphate to 3-buten-2-ol, 3-buten-2-ol to butadiene,1,3-butandediol to 3-buten-2-ol, 1,3-butanediol to 3-hydroxybutyryldiphosphate, 3-hydroxybutyryl phosphate to 3-buten-2-ol, succinyl-CoA tosuccinate, succinyl-CoA to succinate semialdehyde, succinatesemialdehyde to 4-hydroxybutymte, 4-hydroxybutyrate to4-hydroxybutyryl-phosphate, 4-hydroxybutyryl-phosphate to4-hydroxybutyryl-CoA, 4-hydroxybutyryl-CoA to 4-hydroxybutanal,4-hydroxybutanal to 1,4-butanediol, succinate to succinate semialdehyde,succinyl-CoA to 4-hydroxybutyrate, 4-hydroxybutyrate to4-hydroxybutyryl-CoA, 4-hydroxybutyrate to 4-hydroxybutanal,4-hydroxybutyryl-phosphate to 4-hydroxybutanal, 4-hydroxybutyryl-CoA to1,4-butanediol, succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA,3-oxoadipyl-CoA to 3-hydroxyadiply-CoA, 3-hydroxyadiply-CoA to5-carboxy-2-pentenoyl-CoA, 5-carboxy-2-pentenoyl-CoA to adipyl-CoA,adipyl-CoA to adipate, adipyl-CoA to adipate semialdehyde, adipatesemialdehyde to 6-aminocaproate, 6-aminocaproate to caprolactam,6-aminocaproate to 6-aminocaproyl-CoA, 6-aminocaproyl-CoA to6-aminocaproate semialdehyde, 6-aminocaproate semialdehyde tohexamethylenediamine, succinyl-CoA to (R)-methylmalonyl-CoA,(R)-methylmalonyl-CoA to methylmalonate semialdehyde,(R)-methylmalonyl-CoA to (S)-methylmalonyl-CoA, (S)-methylmalonyl-CoA tomethylmalonate semialdehyde, (R)-methylmalonyl-CoA to3-hydroxyisobutyrate, (S)-methylmalonyl-CoA to 3-hydroxyisobutyrate,3-hydroxyisobutyrate to methacrylic acid, 3-hydroxybutyryl-CoA to2-hydroxyisobutyryl-CoA, 2-hydroxyisobutyryl-CoA to 2-hydroxyisobutyricacid, 2-hydroxyisobutyryl-CoA to methacrylyl-CoA, methacrylyl-CoA tomethacrylic acid. One skilled in the art will understand that these aremerely exemplary and that any of the substrate-product pairs disclosedherein suitable to produce a desired product and for which anappropriate activity is available for the conversion of the substrate tothe product can be readily determined by one skilled in the art based onthe teachings herein. Thus, the invention provides a non-naturallyoccurring microbial organism containing at least one exogenous nucleicacid encoding an enzyme or protein, where the enzyme or protein convertsthe substrates and products of an acetyl-CoA or a bioderived compoundpathway, such as that shown in FIG. 1-10.

While generally described herein as a microbial organism that containsan acetyl-CoA or a bioderived compound pathway, it is understood thatthe invention additionally provides a non-naturally occurring microbialorganism comprising at least one exogenous nucleic acid encoding anacetyl-CoA or a bioderived compound pathway enzyme expressed in asufficient amount to produce an intermediate of an acetyl-CoA or abioderived compound pathway. For example, as disclosed herein, anacetyl-CoA or a bioderived compound pathway is exemplified in FIGS.1-10. Therefore, in addition to a microbial organism containing anacetyl-CoA or a bioderived compound pathway that produces acetyl-CoA ora bioderived compound, the invention additionally provides anon-naturally occurring microbial organism comprising at least oneexogenous nucleic acid encoding an acetyl-CoA or a bioderived compoundpathway enzyme, where the microbial organism produces an acetyl-CoA or abioderived compound pathway intermediate, for example, acetate, ACTP,G3P, PYR, Formate, Fald, formyl-CoA, FTHF, Methenyl-THF, Methylene-THF,Glycine, Serine, Methyl-THF, H6P, F6P, DHA, S-hydroxymethyglutathione,S-formylglutathione, OAA, CIT, ICIT, MAL, FUM, AKG, SUCC, SUCCOA,MALCOA, AACOA, 3HBCOA, 3HBALD, 3HB, CROTCOA, CROT, CROALD, CROT-Pi,CROT-PPi, 3-hydroxybutyryl phosphate, 3-hydroxybutyryl diphosphate,succinate semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-phosphate,4-hydroxybutyryl-CoA, 4-hydroxybutanal, 3-oxoadipyl-CoA,3-hydroxyadipyl-CoA, 5-hydroxy-2-pentenoyl-CoA, adipate semialdehyde,6-aminocaproyl-CoA, 6-aminocaproate semialdehyde, (R)-methylmalonyl-CoA,(S)-methylmalonyl-CoA, methylmalonate semialdehyde,3-hydrdoxyisobutyrate, 2-hydroxyisobutyryl-CoA, and methacrylyl-CoA.

It is understood that any of the pathways disclosed herein, as describedin the Examples and exemplified in the Figures, including the pathwaysof FIGS. 1-10, can be utilized to generate a non-naturally occurringmicrobial organism that produces any pathway intermediate or product, asdesired. As disclosed herein, such a microbial organism that produces anintermediate can be used in combination with another microbial organismexpressing downstream pathway enzymes to produce a desired product.However, it is understood that a non-naturally occurring microbialorganism that produces an acetyl-CoA or a bioderived compound pathwayintermediate can be utilized to produce the intermediate as a desiredproduct.

In one embodiment, the invention provides a non-naturally occurringmicrobial organism having an acetyl-CoA pathway, wherein said acetyl-CoApathway comprises a pathway selected from: (1) 1T and 1V; (2) 1T, 1W,and 1X; (3) 1U and 1V; (4) 1U, 1W, and 1X; wherein 1T is afructose-6-phosphate phosphoketolase, wherein 1U is axylulose-5-phosphate phosphoketolase, wherein 1V is aphosphotransacetylase, wherein 1W is an acetate kinase, wherein 1X is anacetyl-CoA transferase, an acetyl-CoA synthetase, or an acetyl-CoAligase; wherein said non-naturally occurring microbial organism furthercomprises a pathway capable of producing a bioderived compound, whereinsaid bioderived compounds is selected from the group consisting of (i)1,4-butanediol or an intermediate thereto, wherein said intermediate isoptionally 4-hydroxybutanoic acid (4-HB); (ii) butadiene (1,3-butadiene)or an intermediate thereto, wherein said intermediate is optionally1,4-butanediol, 1,3-butanediol, 2,3-butanediol, crotyl alcohol,3-buten-2-ol (methyl vinyl carbinol) or 3-buten-1-ol; (iii)1,3-butanediol or an intermediate thereto, wherein said intermediate isoptionally 3-hydroxybutyrate (3-HB), 2,4-pentadienoate, crotyl alcoholor 3-buten-1-ol; (iv) adipate, 6-aminocaproic acid, caprolactam,hexamethylenediamine, levulinic acid or an intermediate thereto, whereinsaid intermediate is optionally adipyl-CoA or 4-aminobutyryl-CoA; (v)methacrylic acid or an ester thereof, 3-hydroxyisobutyrate,2-hydroxyisobutyrate, or an intermediate thereto, wherein said ester isoptionally methyl methacrylate or poly(methyl methacrylate); (vi)1,2-propanediol (propylene glycol), 1,3-propanediol, glycerol, ethyleneglycol, diethylene glycol, triethylene glycol, dipropylene glycol,tripropylene glycol, neopentyl glycol, bisphenol A or an intermediatethereto; (vii) succinic acid or an intermediate thereto; and (viii) afatty alcohol, a fatty aldehyde or a fatty acid comprising C4 to C27carbon atoms, C8 to C18 carbon atoms, C12 to C18 carbon atoms, or C12 toC14 carbon atoms, wherein said fatty alcohol is optionally dodecanol(C12; lauryl alcohol), tridecyl alcohol (C13; 1-tridecanol, tridecanol,isotridecanol), myristyl alcohol (C14; 1-tetradecanol), pentadecylalcohol (C15; 1-pentadecanol, pentadecanol), cetyl alcohol (C16;1-hexadecanol), heptadecyl alcohol (C17; 1-n-heptadecanol, heptadecanol)and stearyl alcohol (C18; 1-octadecanol) or palmitoleyl alcohol (C16unsaturated; cis-9-hexadecen-1-ol). In some aspects, the non-naturallyoccurring microbial organism having an acetyl-CoA pathway can furthercomprise a 1,3-butanediol pathway and an exogenous nucleic acid encodinga 1,3-butanediol pathway enzyme expressed in a sufficient amount toproduce 1,3-butanediol, wherein said 1,3-butanediol pathway comprises apathway selected from: (1) 5A, 5B, 5D, 5E, and 5H; (2) 5A, 5B, 5D, 5F,5G, and 5H; (3) 5C, 5D, 5E, and 5H; (4) 5C, 5D, 5F, 5G, and 5H; (5) 5A,5B, 5D and 5V; and (6) 5C, 5D and 5V, wherein 5A is an acetyl-CoAcarboxylase, wherein 5B is an acetoacetyl-CoA synthase, wherein 5C is anacetyl-CoA:acetyl-CoA acyltransferase, wherein 5D is an acetoacetyl-CoAreductase (ketone reducing), wherein 5E is a 3-hydroxybutyryl-CoAreductase (aldehyde forming), wherein 5F is a 3-hydroxybutyryl-CoAhydrolase, transferase or synthetase, wherein 5G is a 3-hydroxybutyratereductase, wherein 5H is a 3-hydroxybutyraldehyde reductase, wherein 5Vis a 3-hydroxybutyryl-CoA reductase (alcohol forming).

In some aspects, the non-naturally occurring microbial organism havingan acetyl-CoA pathway can further comprise a crotyl alcohol pathway andan exogenous nucleic acid encoding a crotyl alcohol pathway enzymeexpressed in a sufficient amount to produce crotyl alcohol, wherein saidcrotyl alcohol pathway comprises a pathway selected from: (1) 5A, 5B,5D, 5J, 5K, and 5N; (2) 5A, 5B, 5D, 5J, 5L, 5M, and 5N; (3) 5C, 5D, 5J,5K, and 5N; (4) 5C, 5D, 5J, 5L, 5M, and 5N; (5) 5A, 5B, 5D, 5J and 5U;and (6) 5C, 5D, 5J and 5U, wherein 5A is an acetyl-CoA carboxylase,wherein 5B is an acetoacetyl-CoA synthase, wherein 5C is anacetyl-CoA:acetyl-CoA acyltransferase, wherein 5D is an acetoacetyl-CoAreductase (ketone reducing), wherein 5J is a 3-hydroxybutyryl-CoAdehydratase, wherein 5K is a crotonyl-CoA reductase (aldehyde forming),wherein 5L is a crotonyl-CoA hydrolase, crotonyl-CoA transferase orcrotonyl-CoA synthetase, wherein 5M is a crotonate reductase, wherein 5Nis a crotonaldehyde reductase, wherein 5U is a crotonyl-CoA reductase(alcohol forming).

In some aspects, the non-naturally occurring microbial organism havingan acetyl-CoA pathway can further comprise a butadiene pathway and anexogenous nucleic acid encoding a butadiene pathway enzyme expressed ina sufficient amount to produce butadiene, wherein said butadiene pathwaycomprises a pathway selected from: (1) 5A, 5B, 5D, 5E, 5H, 6A, 6B, 6C,and 6G; (2) 5A, 5B, 5D, 5F, 5G, 5H, 6A, 6B, 6C, and 6G; (3) 5C, 5D, 5E,5H, 6A, 6B, 6C, and 6G; (4) 5C, 5D, 5F, 5G, 5H, 6A, 6B, 6C, and 6G; (5)5A, 5B, 5D, 5E, 5H, 6A, 6F, and 6G; (6) 5A, 5B, 5D, 5F, 5G, 5H, 6A, 6F,and 6G; (7) 5C, 5D, 5E, 5H, 6A, 6F, and 6G; (8) 5C, 5D, 5F, 5G, 5H, 6A,6F, and 6G; (9) 5A, 5B, 5D, 5E, 5H, 6E, 6C, and 6G; (10) 5A, 5B, 5D, 5F,5G, 5H, 6E, 6C, and 6G; (11) 5C, 5D, 5E, 5H, 6E, 6C, and 6G; (12) 5C,5D, 5F, 5G, 5H, 6E, 6C, and 6G; (13) 5A, 5B, 5D, 5E, 5H, 6D, and 6G;(14) 5A, 5B, 5D, 5F, 5G, 5H, 6D, and 6G; (15) 5C, 5D, 5E, 5H, 6D, and6G; (16) 5C, 5D, 5F, 5G, 5H, 6D, and 6G; (17) 5A, 5B, 5D, 5J, 5K, 5N,and 5S; (18) 5A, 5B, 5D, 5J, 5L, 5M, 5N, and 5S; (19) 5C, 5D, 5J, 5K,5N, and 5S; (20) 5C, 5D, 5J, 5L, 5M, 5N, and 5S; (21) 5A, 5B, 5D, 5J,5K, 5N, 5R, and 5Q; (22) 5A, 5B, 5D, 5J, 5L, 5M, 5N, 5R, and 5Q; (23)5C, 5D, 5J, 5K, 5N, 5R, and 5Q; (24) 5C, 5D, 5J, 5L, 5M, 5N, 5R, and 5Q;(25) 5A, 5B, 5D, 5J, 5K, 5N, 5O, 5P, and 5Q; (26) 5A, 5B, 5D, 5J, 5L,5M, 5N, 5O, 5P, and 5Q; (27) 5C, 5D, 5J, 5K, 5N, 5O, 5P, and 5Q; (28)5C, 5D, 5J, 5L, 5M, 5N, 5O, 5P, and 5Q; (29) 5A, 5B, 5D, 5J, 5K, 5N, 5O,and 5T; (30) 5A, 5B, 5D, 5J, 5L, 5M, 5N, 5O, and 5T; (31) 5C, 5D, 5J,5K, 5N, 5O, and 5T; (32) 5C, 5D, 5J, 5L, 5M, 5N, 5O, and 5T; (33) 5A,5B, 5D, 5V, 6A, 6B, 6C, and 6G; (34) 5C, 5D, 5V, 6A, 6B, 6C, and 6G;(35) 5A, 5B, 5D, 5J, 5U, and 5S; (36) 5C, 5D, 5J, 5U, and 5S; (37) 5A,5B, 5D, 5J, 5U, 5R, and 5Q; (38) 5C, 5D, 5J, 5U, 5R, and 5Q; (39) 5A,5B, 5D, 5J, 5U, 5O, 5P, and 5Q; (40) 5C, 5D, 5J, 5U, 5O, 5P, and 5Q;(41) 5A, 5B, 5D, 5J, 5U, 50, and 5T; and (42) 5C, 5D, 5J, 5U, 5O, and5T, wherein 5A is an acetyl-CoA carboxylase, wherein 5B is anacetoacetyl-CoA synthase, wherein 5C is an acetyl-CoA:acetyl-CoAacyltransferase, wherein 5D is an acetoacetyl-CoA reductase (ketonereducing), wherein 5E is a 3-hydroxybutyryl-CoA reductase (aldehydeforming), wherein 5F is a 3-hydroxybutyryl-CoA hydrolase,3-hydroxybutyryl-CoA transferase or 3-hydroxybutyryl-CoA synthetase,wherein 5G is a 3-hydroxybutyrate reductase, wherein 5H is a3-hydroxybutyraldehyde reductase, wherein 5J is a 3-hydroxybutyryl-CoAdehydratase, wherein 5K is a crotonyl-CoA reductase (aldehyde forming),wherein 5L is a crotonyl-CoA hydrolase, crotonyl-CoA transferase orcrotonyl-CoA synthetase, wherein 5M is a crotonate reductase, wherein 5Nis a crotonaldehyde reductase, wherein 50 is a crotyl alcohol kinase,wherein 5P is a 2-butenyl-4-phosphate kinase, wherein 5Q is a butadienesynthase, wherein 5R is a crotyl alcohol diphosphokinase, wherein 5S ischemical dehydration or a crotyl alcohol dehydratase, wherein 5T is abutadiene synthase (monophosphate), wherein 5U is a crotonyl-CoAreductase (alcohol forming), wherein 5V is a 3-hydroxybutyryl-CoAreductase (alcohol forming), wherein 6A is a 1,3-butanediol kinase,wherein 6B is a 3-hydroxybutyrylphosphate kinase, wherein 6C is a3-hydroxybutyryldiphosphate lyase, wherein 6D is a 1,3-butanedioldiphosphokinase, wherein 6E is a 1,3-butanediol dehydratase, wherein 6Fis a 3-hydroxybutyrylphosphate lyase, wherein 6G is a 3-buten-2-oldehydratase or chemical dehydration.

In some aspects, the non-naturally occurring microbial organism havingan acetyl-CoA pathway can further comprise a 3-buten-2-ol pathway and anexogenous nucleic acid encoding a 3-buten-2-ol pathway enzyme expressedin a sufficient amount to produce 3-buten-2-ol, wherein said3-buten-2-ol pathway comprises a pathway selected from: (1) 5A, 5B, 5D,5E, 5H, 6A, 6B, and 6C; (2) 5A, 5B, 5D, 5F, 5G, 5H, 6A, 6B, and 6C; (3)5C, 5D, 5E, 5H, 6A, 6B, and 6C; (4) 5C, 5D, 5F, 5G, 5H, 6A, 6B, and 6C;(5) 5A, 5B, 5D, 5E, 5H, 6A, and 6F; (6) 5A, 5B, 5D, 5F, 5G, 5H, 6A, and6F; (7) 5C, 5D, 5E, 5H, 6A, and 6F; (8) 5C, 5D, 5F, 5G, 5H, 6A, and 6F;(9) 5A, 5B, 5D, 5E, 5H, 6E, and 6C; (10) 5A, 5B, 5D, 5F, 5G, 5H, 6E, and6C; (11) 5C, 5D, 5E, 5H, 6E, and 6C; (12) 5C, 5D, 5F, 5G, 5H, 6E, and6C; (13) 5A, 5B, 5D, 5E, 5H, and 6D; (14) 5A, 5B, 5D, 5F, 5G, 5H, and6D; (15) 5C, 5D, 5E, 5H, and 6D; (16) 5C, 5D, 5F, 5G, 5H, and 6D; (17)5A, 5B, 5D, 5V, 6A, 6B, and 6C; (18) 5C, 5D, 5V, 6A, 6B, and 6C; (19)5A, 5B, 5D, 5V, 6A, and 6F; (20) 5C, 5D, 5V, 6A, and 6F; (21) 5A, 5B,5D, 5V, 6E, and 6C; (22) 5C, 5D, 5V, 6E, and 6C; (23) 5A, 5B, 5D, 5V and6D; and (24) 5C, 5D, 5V and 6D, wherein 5A is an acetyl-CoA carboxylase,wherein 5B is an acetoacetyl-CoA synthase, wherein 5C is anacetyl-CoA:acetyl-CoA acyltransferase, wherein 5D is an acetoacetyl-CoAreductase (ketone reducing), wherein 5E is a 3-hydroxybutyryl-CoAreductase (aldehyde forming), wherein 5F is a 3-hydroxybutyryl-CoAhydrolase, 3-hydroxybutyryl-CoA transferase or 3-hydroxybutyryl-CoAsynthetase, wherein 5G is a 3-hydroxybutyrate reductase, wherein 5H is a3-hydroxybutyraldehyde reductase, wherein 5V is a 3-hydroxybutyryl-CoAreductase (alcohol forming), wherein 6A is a 1,3-butanediol kinase,wherein 6B is a 3-hydroxybutyrylphosphate kinase, wherein 6C is a3-hydroxybutyryldiphosphate lyase, wherein 6D is a 1,3-butanedioldiphosphokinase, wherein 6E is a 1,3-butanediol dehydratase, wherein 6Fis a 3-hydroxybutyrylphosphate lyase.

In some aspects the non-naturally occurring microbial organism having anacetyl-CoA pathway can further comprise a 1,4-butanediol pathway and anexogenous nucleic acid encoding a 1,4-butanediol pathway enzymeexpressed in a sufficient amount to produce 1,4-butanediol, wherein said1,4-butanediol pathway comprises a pathway selected from: (1) 7B, 7C,7D, 7E, 7F, and 7G; (2) 7A, 7H, 7C, 7D, 7E, 7F, and 7G; (3) 7I, 7D, 7E,7F, and 7G; (4) 7B, 7C, 7K, and 7G; (5) 7A, 7H, 7C, 7K, and 7G; (6) 7I,7K, and 7G; (7) 7B, 7C, 7D, 7L, and 7G; (8) 7A, 7H, 7C, 7D, 7L, and 7G;(9) 7I, 7D, 7L, and 7G; (10) 7B, 7C, 7J, 7F, and 7G; (11) 7A, 7H, 7C,7J, 7F, and 7G; (12) 7I, 7J, 7F, and 7G; (13) 7B, 7C, 7D, 7E, and 7M;(14) 7A, 7H, 7C, 7D, 7E, and 7M; and (15) 7I, 7D, 7E, and 7M, wherein 7Ais a succinyl-CoA transferase or a succinyl-CoA synthetase, wherein 7Bis a succinyl-CoA reductase (aldehyde forming), wherein 7C is a 4-HBdehydrogenase, wherein 7D is a 4-HB kinase, wherein 7E is aphosphotrans-4-hydroxybutyrylase, wherein 7F is a 4-hydroxybutyryl-CoAreductase (aldehyde forming), wherein 7G is a 1,4-butanedioldehydrogenase, wherein 7H is a succinate reductase, wherein 71 is asuccinyl-CoA reductase (alcohol forming), wherein 7J is a4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA synthetase,wherein 7K is a 4-HB reductase, wherein 7L is a4-hydroxybutyryl-phosphate reductase, wherein 7M is a4-hydroxybutyryl-CoA reductase (alcohol forming).

In some aspects, the non-naturally occurring microbial organism havingan acetyl-CoA pathway can further comprise an adipate pathway and anexogenous nucleic acid encoding an adipate pathway enzyme expressed in asufficient amount to produce adipate, wherein said adipate pathwaycomprises 8A, 8B, 8C, 8D and 8L, wherein 8A is a 3-oxoadipyl-CoAthiolase, wherein 8B is a 3-oxoadipyl-CoA reductase, wherein 8C is a3-hydroxyadipyl-CoA dehydratase, wherein 8D is a5-carboxy-2-pentenoyl-CoA reductase, wherein 8L is an adipyl-CoAhydrolase, adipyl-CoA ligase, adipyl-CoA transferase, orphosphotransadipylase/adipate kinase.

In some aspects, the non-naturally occurring microbial organism havingan acetyl-CoA pathway can further comprise a 6-aminocaproate pathway andan exogenous nucleic acid encoding a 6-aminocaproate pathway enzymeexpressed in a sufficient amount to produce 6-aminocaproate, whereinsaid 6-aminocaproate pathway comprises 8A, 8B, 8C, 8D, 8E, and 8F,wherein 8A is a 3-oxoadipyl-CoA thiolase, wherein 8B is a3-oxoadipyl-CoA reductase, wherein 8C is a 3-hydroxyadipyl-CoAdehydratase, wherein 8D is a 5-carboxy-2-pentenoyl-CoA reductase,wherein 8E is an adipyl-CoA reductase (aldehyde forming), wherein 8F isa 6-aminocaproate transaminase or 6-aminocaproate dehydrogenase.

In some aspects, the non-naturally occurring microbial organism havingan acetyl-CoA pathway can further comprise a caprolactam pathway and anexogenous nucleic acid encoding a caprolactam pathway enzyme expressedin a sufficient amount to produce caprolactam, wherein said caprolactampathway comprises: (1) 8A, 8B, 8C, 8D, 8E, 8F, and 8H; or (2) 8A, 8B,8C, 8D, 8E, 8F, 8G, and 8I, wherein 8A is a 3-oxoadipyl-CoA thiolase,wherein 8B is a 3-oxoadipyl-CoA reductase, wherein 8C is a3-hydroxyadipyl-CoA dehydratase, wherein 8D is a5-carboxy-2-pentenoyl-CoA reductase, wherein 8E is an adipyl-CoAreductase (aldehyde forming), wherein 8F is a 6-aminocaproatetransaminase or 6-aminocaproate dehydrogenase, wherein 8G is a6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoA synthase,wherein 8H is an amidohydrolase, wherein 81 is spontaneous cyclization.

In some aspects, the non-naturally occurring microbial organism havingan acetyl-CoA pathway can further comprise a hexamethylenediaminepathway and an exogenous nucleic acid encoding a hexamethylenediaminepathway enzyme expressed in a sufficient amount to producehexamethylenediamine, wherein said hexamethylenediamine pathwaycomprises 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8J, 8K, wherein 8A is a3-oxoadipyl-CoA thiolase, wherein 8B is a 3-oxoadipyl-CoA reductase,wherein 8C is a 3-hydroxyadipyl-CoA dehydratase, wherein 8D is a5-carboxy-2-pentenoyl-CoA reductase, wherein 8E is an adipyl-CoAreductase (aldehyde forming), wherein 8F is a 6-aminocaproatetransaminase or 6-aminocaproate dehydrogenase, wherein 8G is a6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoA synthase,wherein 8J is a 6-aminocaproyl-CoA reductase (aldehyde forming), wherein8K is a hexamethylenediamine transaminase or hexamethylenediaminedehydrogenase.

In some aspects, the non-naturally occurring microbial organism havingan acetyl-CoA pathway can further comprise a methacrylic acid pathwayand an exogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, whereinsaid methacrylic acid pathway comprises a pathway selected from: (1) 9A,9B, 9C, 9D, and 9E; (2) 9A, 9F, and 9E; (3) 9A, 9B, 9F, and 9E; (4) 9A,9C, 9D, and 9E; and (5) 10A, 10B, 10C, 10D, and 10E, wherein 9A is amethylmalonyl-CoA mutase, wherein 9B is a methylmalonyl-CoA epimerase,wherein 9C is a methylmalonyl-CoA reductase (aldehyde forming), wherein9D is a methylmalonate semialdehyde reductase, wherein 9E is a3-hydroxyisobutyrate dehydratase, wherein 9F is a methylmalonyl-CoAreductase (alcohol forming), wherein 10A is an acetyl-CoA:acetyl-CoAacyltransferase, wherein 10B is an acetoacetyl-CoA reductase (ketonereducing), wherein 10C is a 3-hydroxybutyrl-CoA mutase, wherein 10D is a2-hydroxyisobutyryl-CoA dehydratase, wherein 10E is a methacrylyl-CoAsynthetase, methacrylyl-CoA hydrolase, or methacrylyl-CoA transferase.

In some aspects, the non-naturally occurring microbial organism havingan acetyl-CoA pathway can further comprise a 2-hydroxyisobutyric acidpathway and an exogenous nucleic acid encoding a 2-hydroxyisobutyricacid pathway enzyme expressed in a sufficient amount to produce2-hydroxyisobutyric acid, wherein said 2-hydroxyisobutyric acid pathwaycomprises 10A, 10B, 10C, and 10F, wherein 10A is anacetyl-CoA:acetyl-CoA acyltransferase, wherein 10B is an acetoacetyl-CoAreductase (ketone reducing), wherein 10C is a 3-hydroxybutyrl-CoAmutase, wherein 10F is a 2-hydroxyisobutyryl-CoA synthetase,2-hydroxyisobutyryl-CoA hydrolase, or 2-hydroxyisobutyryl-CoAtransferase.

In certain embodiments, such as the above non-naturally occurringmicrobial organisms having an acetyl-CoA pathway and 1,4-butanediolpathway, an adipate pathway, a 6-aminocaproate pathway, a caprolactampathway, a hexamethylenediamine pathway or a methacrylic acid pathway,the non-naturally occurring microbial organism can further comprise asuccinyl-CoA pathway and an exogenous nucleic acid encoding asuccinyl-CoA pathway enzyme expressed in a sufficient amount to producesuccinyl-CoA, wherein said succinyl-CoA pathway comprises a pathwayselected from: (1) 4H, 4I, 4J, and 4K; (2) 4H, 4I, 4N, and 4E; (3) 4H,4I, 4N, 4C, 4D, and 4E; (4) 4L, 4H, 4I, 4J, and 4K; (5) 4L, 4H, 4I, 4N,and 4E; (6) 4L, 4H, 4I, 4N, 4C, 4D, and 4E; (7) 4A, 4H, 4I, 4J, and 4K;(8) 4A, 4H, 4I, 4N, and 4E; (9) 4A, 4H, 4I, 4N, 4C, 4D, and 4E; (10) 4M,4C, 4D, and 4E; (11) 4F, 4L, 4H, 4I, 4J, and 4K; (12) 4F, 4L, 4H, 4I,4N, and 4E; (13) 4F, 4L, 4H, 4I, 4N, 4C, 4D, and 4E; (14) 4F, 4M, 4C,4D, and 4E; (15) 4F, 4G, 4H, 4I, 4J, and 4K; (16) 4F, 4G, 4H, 4I, 4N,and 4E; and (17) 4F, 4G, 4H, 4I, 4N, 4C, 4D, and 4E, wherein 4A is a PEPcarboxylase or PEP carboxykinase, wherein 4B is a malate dehydrogenase,wherein 4C is a fumarase, wherein 4D is a fumarate reductase, wherein 4Eis a succinyl-CoA synthetase or succinyl-CoA transferase, wherein 4F isa pyruvate kinase or PTS-dependent substrate import, wherein 4G is apyruvate dehydrogenase, pyruvate formate lyase, or pyruvate:ferredoxinoxidoreductase, wherein 4H is a citrate synthase, wherein 41 is anaconitase, wherein 4J is an isocitrate dehydrogenase, wherein 4K is analpha-ketoglutarate dehydrogenase, wherein 4L is a pyruvate carboxylase,wherein 4M is a malic enzyme, wherein 4N is an isocitrate lyase andmalate synthase.

In certain aspects, the non-naturally occurring microbial organismhaving an acetyl-CoA pathway can be a microbial organism speciesselected from a bacteria, yeast, or fungus.

The invention further provides non-naturally occurring microbialorganisms that have elevated or enhanced synthesis or yields ofacetyl-CoA or a bioderived compound and methods of using thosenon-naturally occurring organisms to produce such biosynthetic products,the bioderived compound including alcohols, diols, fatty acids, glycols,organic acids, alkenes, dienes, organic amines, organic aldehydes,vitamins, nutraceuticals and pharmaceuticals. The enhanced synthesis ofintracellular acetyl-CoA enables enhanced production of bioderivedcompounds for which acetyl-CoA is an intermediate and further, may havebeen rate-limiting.

The non-naturally occurring microbial organisms having enhanced yieldsof a biosynthetic product include one or more of the various pathwayconfigurations employing a methanol dehydrogenase for methanoloxidation, a formaldehyde fixation pathway and/or an acetyl-CoAenhancing pathway, e.g. phosphoketolase, for directing the carbon frommethanol into acetyl-CoA and other desired products via formaldehydefixation. The various different methanol oxidation and formaldehydefixation configurations exemplified below can be engineered inconjunction with any or each of the various methanol oxidation,formaldehyde fixation, formate reutilization, acetyl-CoA or bioderivedcompound pathways exemplified previously and herein. The metabolicmodifications exemplified below increase biosynthetic product yieldsover, for example, endogenous methanol utilization pathways because theyfurther focus methanol derived carbon into the assimilation pathwaysdescribed herein, decrease inefficient use of methanol carbon throughcompeting methanol utilization and/or formaldehyde fixation pathwaysand/or increase the production of reducing equivalents.

In this regard, methylotrophs microbial organisms utilize methanol asthe sole source of carbon and energy. In such methylotrophic organisms,the oxidation of methanol to formaldehyde is catalyzed by one of threedifferent enzymes: NADH dependent methanol dehydrogenase (MeDH),PQQ-dependent methanol dehydrogenase (MeDH-PQQ) and alcohol oxidase(AOX). Methanol oxidase is a specific type of AOX with activity onmethanol. Gram positive bacterial methylotrophs such as Bacillusmethanolicus utilize a cytosolic MeDH which generates reducingequivalents in the form ofNADH. Gram negative bacterial methylotrophsutilize periplasmic PQQ-containing methanol dehydrogenase enzymes whichtransfer electrons from methanol to specialized cytochromes CL, andsubsequently to a cytochrome oxidase (Afolabi et al, Biochem40:9799-9809 (2001)). Eukaryotic methylotrophs employ a peroxisomaloxygen-consuming and hydrogen-peroxide producing alcohol oxidase.

Bacterial methylotrophs are found in in the genera Bacillus,Methylobacterium, Methyloversatilis, Methylococcus, Methylocystis andHyphomicrobium. These organisms utilize either the serine cycle (type orthe RuMP cycle (type I) to further assimilate formaldehyde into centralmetabolism (Hanson and Hanson, Microbiol Rev 60:439-471 (1996)). Asdescribed previously, the RuMP pathway combines formaldehyde withribulose monophosphate to form hexulose-6-phosphate, which is furtherconverted to fructose-6-phosphate (see FIG. 1, step C). In the serinecycle formaldehyde is initially converted to 5,10-methylene-THF, whichis combined with glycine to form serine. Overall, the reactions of theserine cycle produce one equivalent of acetyl-CoA from three equivalentsof methanol (Anthony, Science Prog 94:109-37 (2011)). The RuMP cyclealso yields one equivalent of acetyl-CoA from three equivalents methanolin the absence of phosphoketolase activity or a formate assimilationpathway. Genetic tools are available for numerous prokaryoticmethylotrophs and methanotrophs.

Eukaryotic methylotrophs are found in the genera Candida, Pichia,Ogataea, Kuraishia and Komagataella. Particularly useful methylotrophichost organisms are those with well-characterized genetic tools and geneexpression systems such as Hansenula polymorpha, Pichia pastor's,Candida boidinii and Pichia methanolica (for review see Yurimoto et al,Int J Microbiol (2011)). The initial step of methanol assimilation ineukaryotic methylotrophs occurs in the peroxisomes, where methanol andoxygen are oxidized to formaldehyde and hydrogen peroxide by alcoholoxidase (AOX). Formaldehyde assimilation with xylulose-5-phosphate viaDHA synthase also occurs in the peroxisomes. During growth on methanol,the two enzymes DHA synthase and AOX together comprise 80% of the totalcell protein (Horiguchi et al, J Bacteriol 183:6372-83 (2001)). DHAsynthase products, DHA and glyceraldehyde-3-phosphate, are secreted intothe cytosol where they undergo a series of rearrangements catalyzed bypentose phosphate pathway enzymes, and are ultimately converted tocellular constituents and xylulose-5-phosphate, which is transportedback into the peroxisomes. The initial step of formaldehydedissimilation, catalyzed by S-(hydroxymethyl)-glutathione synthase, alsooccurs in the peroxisomes. Like the bacterial methylotrophic pathwaysdescribed above, eukaryotic methylotrophic pathways convert threeequivalents of methanol to at most one equivalent of acetyl-CoA becausethey lack phosphoketolase activity or a formate assimilation pathway.

As exemplified further below, the various configurations of metabolicmodifications disclosed herein for enhancing product yields via methanolderived carbon include enhancing methanol oxidation and production ofreducing equivalents using either and an endogenous NADH dependentmethanol dehydrogenase, an exogenous NADH dependent methanoldehydrogenase, both an endogenous NADH dependent methanol dehydrogenaseand exogenous NADH dependent methanol dehydrogenase alone or incombination with one or more metabolic modifications that attenuate, forexample, DHA synthase and/or AOX. In addition, other metabolicmodifications as exemplified below that reduce carbon flux away frommethanol oxidation and formaldehyde fixation also can be included, aloneor in combination, with the methanol oxidation and formaldehyde fixationpathway configurations disclosed herein that enhance carbon flux intoproduct precursors such as acetyl-CoA and, therefore, enhance productyields.

Accordingly, the microbial organism of the invention having one or moreof any of the above and/or below metabolic modifications to a methanolutilization pathway and/or formaldehyde assimilation pathwayconfigurations for enhancing product yields can be combined with any oneor more, including all of the previously described methanol oxidation,formaldehyde fixation, formate reutilization, and/or acetyl-CoA pathwaysto enhance the yield and/or production of a product such as any of thebioderived compounds described herein.

Given the teachings and guidance provided herein, the methanol oxidationand formaldehyde fixation pathway configurations can be equallyengineered into both prokaryotic and eukaryotic organisms. Inprokaryotic microbial organisms, for example, one skilled in the artwill understand that utilization of an endogenous methanol oxidationpathway enzyme or expression of an exogenous nucleic acid encoding amethanol oxidation pathway enzyme will naturally occur cytosolicallybecause prokaryotic organisms lack peroxisomes. In eukaryotic microbialorganisms one skilled in the art will understand that certain methanoloxidation pathways occur in the peroxisome as described above and thatcytosolic expression of the methanol oxidation pathway or pathwaysdescribed herein to enhance product yields can be beneficial. Theperoxisome located pathways and competing pathways remain or,alternatively, attenuated as described below to further enhance methanoloxidation and formaldehyde fixation.

With respect to eukaryotic microbial host organisms, those skilled inthe art will know that yeasts and other eukaryotic microorganismsexhibit certain characteristics distinct from prokaryotic microbialorganisms. When such characteristics are desirable, one skilled in theart can choose to use such eukaryotic microbial organisms as a host forengineering the various different methanol oxidation and formaldehydefixation configurations exemplified herein for enhancing product yields.For example, yeast are robust organisms, able to grow over a wide pHrange and able to tolerate more impurities in the feedstock. Yeast alsoferment under low growth conditions and are not susceptible to infectionby phage. Less stringent aseptic design requirements can also reduceproduction costs. Cell removal, disposal and propagation are alsocheaper, with the added potential for by-product value for animal feedapplications. The potential for cell recycle and semi-continuousfermentation offers benefits in increased overall yields and rates.Other benefits include: potential for extended fermentation times underlow growth conditions, lower viscosity broth (vs E. coli) with insolublehydrophobic products, the ability to employ large fermenters withexternal loop heat exchangers.

Eukaryotic host microbial organisms suitable for engineering carbonefficient methanol utilization capability can be selected from, and thenon-naturally occurring microbial organisms generated in, for example,yeast, fungus or any of a variety of other microorganisms applicable tofermentation processes. As described previously, exemplary yeasts orfungi include species selected from the genera Saccharomyces,Schizosaccharomyces, Schizochytrium, Rhodotorula, Thraustochytrium,Aspergillus, Kluyveromyces, Issatchenkia, Yarrowia, Candida, Pichia,Ogataea, Kuraishia, Hansenula and Komagataella. Useful host organismsinclude Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenulapolymorpha, Pichia methanolica, Candida boidinii, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichiapastoris, Rhizopus arrhizus, Rhizopus oryzae, Yarrowia lipolytica,Issatchenkia orientalis and the like.

The methanol oxidation and/or formaldehyde assimilation pathwayconfigurations described herein for enhancing product yields include,for example, a NADH-dependent methanol dehydrogenase (MeDH), one or moreformaldehyde assimilation pathways and/or one or more phosphoketolases.Such engineered pathways provide a yield advantage over endogenouspathways found in methylotrophic organisms. For example, methanolassimilation via methanol dehydrogenase provides reducing equivalents inthe useful form of NADH, whereas alcohol oxidase and PQQ-dependentmethanol dehydrogenase do not. Several product pathways described hereinhave several NADH-dependent enzymatic steps. In addition, deletion ofredox-inefficient methanol oxidation enzymes as described further below,combined with increased cytosolic or peroxisomal expression of anNADH-dependent methanol dehydrogenase, improves the ability of theorganism to extract useful reducing equivalents from methanol. In someaspects, if NADH-dependent methanol dehydrogenase is engineered into theperoxisome, an efficient means of shuttling redox in the form of NADHout of the peroxisome and into the cytosol can be included. Furtheremployment of a formaldehyde assimilation pathway in combination with aphosphoketolase or formate assimilation pathway enables high yieldconversion of methanol to acetyl-CoA, and subsequently to acetyl-CoAderived products.

For example, in a eukaryotic organism such as Pichia pastoris, deletingthe endogenous alcohol oxidase and peroxisomal formaldehyde assimilationand dissimilation pathways, and expressing redox and carbon-efficientcytosolic methanol utilization pathways significantly improves the yieldof dodecanol, an acetyl-CoA derived product. The maximum docidecanolyield of Pichia pastoris from methanol using endogenous methanol oxidaseand formaldehyde assimilation enzymes is 0.256 g dodecanol/g methanol.Adding one or more heterologous cytosolic phosphoketolase enzymes, incombination with a formaldehyde assimilation pathway such as the DHApathway or the RUMP pathway, boosts the dodecanol yield to 0.306 gdodecanol/g methanol. Deletion of peroxisomal methanol oxidase andformaldehyde assimilation pathway enzymes (alcohol oxidase, DHAsynthase), and replacement with cytosolic methanol dehydrogenase (NADHdependent) and formaldehyde assimilation pathways, together with aphosphoketolase, provides a significant boost of yield to 0.422 g/g.

Strain design (assumes DHA pathway) Max FA yield (g dodecanol/g MeOH)Pichia + AOX + fatty acid pathway 0.256 Pichia + AOX + PK 0.306 Pichia +MeDH + PK 0.422

The combination of NADH-dependent methanol dehydrogenase andphosphoketolase together results in a significant boost in yield forother acetyl-CoA derived products. For 13-BDO production as shown viathe pathway in FIG. 5, methanol dehydrogenase in combination withphosphoketolase improves the yield from 0.469 to 0.703 g 13-BDO/gmethanol.

Strain design (assumes RuMP pathway) MeOH per 1,3-BDO (mol/mol) 13-BDOper MeOH (g/g) 13-BDO + AOX 6 .469 13-BDO + AOX + PK 5.778 .487 13-BDO +MeDH + PK 4 .703

Metabolic modifications for enabling redox- and carbon-efficientcytosolic methanol utilization in a eukaryotic or prokaryotic organismare exemplified in further detail below.

In one embodiment, the invention provides cytosolic expression of one ormore methanol oxidation and/or formaldehyde assimilation pathwaysEngineering into a host microbial organism carbon- and redox-efficientcytosolic formaldehyde assimilation can be achieved by expression of oneor more endogenous or exogenous methanol oxidation pathways and/or oneor more endogenous or exogenous formaldehyde assimilation pathwayenzymes in the cytosol. An exemplary pathway for methanol oxidationincludes NADH dependent methanol dehydrogenase as shown in FIG. 1.Exemplary pathways for converting cytosolic formaldehyde into glycolyticintermediates also are shown in FIG. 1. Such pathways include methanoloxidation via expression of an cytosolic NADH dependent methanoldehydrogenase, formaldehyde fixation via expression of cytosolic DHAsynthase, both methanol oxidation via expression of an cytosolic NADHdependent methanol dehydrogenase and formaldehyde fixation viaexpression of cytosolic DHA synthase alone or together with themetabolic modifications exemplified below that attenuate less beneficialmethanol oxidation and/or formaldehyde fixation pathways. Suchattenuating metabolic modifications include, for example, attenuation ofalcohol oxidase, attenuation of DHA kinase and/or when utilization ofribulose-5-phosphate (Ru5P) pathway for formaldehyde fixationattenuation of DHA synthase.

For example, in the carbon-efficient DHA pathway of formaldehydeassimilation shown in FIG. 1, step D, formaldehyde is converted todihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (GAP) by DHAsynthase (FIG. 1D). DHA and G3P are then converted tofructose-6-phosphate in one step by F6P aldolase (FIG. 1C) or in threesteps by DHA kinase, FBP aldolase and fructose-1,6-bisphosphatase (notshown). Formation of F6P from DHA and G3P by F6P aldolase is more ATPefficient than using DHA kinase, FBP aldolase, andfructose-1,6-bisphosphatase. Rearrangement of F6P and E4P by enzymes ofthe pentose phosphate pathway (transaldolase, transketolase, R5Pepimerase and Ru5P epimerase) regenerates xylulose-5-phosphate, the DHAsynthase substrate. Conversion of F6P to acetyl-phosphate and E4P (FIG.1T), or Xu5P to G3P and acetyl-phosphate (FIGS. 1T and 1U) by one ormore phosphoketolase enzymes results in the carbon-efficient generationof cytosolic acetyl-CoA. Exemplary enzymes catalyzing each step of thecarbon efficient DHA pathway are described elsewhere herein.

An alternate carbon efficient pathway for formaldehyde assimilationproceeding through ribulose-5-phosphate (Ru5P) is shown in FIG. 1, stepB. The formaldehyde assimilation enzyme of this pathway is3-hexulose-6-phosphate synthase, which combines ru5p and formaldehyde toform hexulose-6-phosphate (FIG. 1B). 6-Phospho-3-hexuloisomeraseconverts H6P to F6P (FIG. 1C). Regeneration of Ru5P from F6P proceeds bypentose phosphate pathway enzymes. Carbon-efficient phosphoketolaseenzymes catalyze the conversion of F6P and/or Xu5P to acetyl-phosphateand pentose phosphate intermediates. Exemplary enzymes catalyzing eachstep of the carbon efficient RuMP pathway are described elsewhereherein. Yet another approach is to combine the RuMP and DHA pathwaystogether (FIG. 1).

Thus, in this embodiment, conversion of cytosolic formaldehyde intoglycolytic intermediates can occur via expression of a cytosolic3-hexulose-6-phosphate (3-Hu6P) synthase and6-phospho-3-hexuloisomerase. Thus, exemplary pathways that can beengineered into a microbial organism of the invention can includemethanol oxidation via expression of a cytosolic NADH dependent methanoldehydrogenase, formaldehyde fixation via expression of cytosolic 3-Hu6Psynthase and 6-phospho-3-hexuloisomerase, both methanol oxidation viaexpression of an cytosolic NADH dependent methanol dehydrogenase andformaldehyde fixation via expression of cytosolic 3-Hu6P synthase and6-phospho-3-hexuloisomerase alone or together with the metabolicmodifications exemplified below that attenuate less beneficial methanoloxidation and/or formaldehyde fixation pathways. Such attenuatingmetabolic modifications include, for example, attenuation of alcoholoxidase, attenuation of DHA kinase and/or attenuation of DHA kinaseand/or attenuation of DHA synthase (e.g. when ribulose-5-phosphate(Ru5P) pathway for formaldehyde fixation is utilized).

In yet another embodiment increased product yields can be accomplishedby engineering into the host microbial organism of the invention boththe RuMP and DHA pathways as shown in FIG. 1. In this embodiment, themicrobial organisms can have cytosolic expression of one or moremethanol oxidation and/or formaldehyde assimilation pathways. Theformaldehyde assimilation pathways can include both assimilation throughcytosolic DHA synthase and 3-Hu6P synthase. Such pathways includemethanol oxidation via expression of a cytosolic NADH dependent methanoldehydrogenase, formaldehyde fixation via expression of cytosolic DHAsynthase and 3-Hu6P synthase, both methanol oxidation via expression ofan cytosolic NADH dependent methanol dehydrogenase and formaldehydefixation via expression of cytosolic DHA synthase and 3-Hu6P synthasealone or together with the metabolic modifications exemplifiedpreviously and also below that attenuate less beneficial methanoloxidation and/or formaldehyde fixation pathways. Such attenuatingmetabolic modifications include, for example, attenuation of alcoholoxidase, attenuation of DHA kinase and/or attenuation of DHA kinaseand/or attenuation of DHA synthase (e.g. when ribulose-5-phosphate(Ru5P) pathway for formaldehyde fixation is utilized).

Increasing the expression and/or activity of one or more formaldehydeassimilation pathway enzymes in the cytosol can be utilized toassimilate formaldehyde at a high rate. Increased activity can beachieved by increased expression, altering the ribosome binding site,altering the enzyme activity, or altering the sequence of the gene toensure, for example, that codon usage is balanced with the needs of thehost organism, or that the enzyme is targeted to the cytosol asdisclosed herein.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism as described herein, wherein the microbial organismfurther includes attenuation of one or more endogenous enzymes, whichenhances carbon flux through acetyl-CoA. For example, in some aspects,the endogenous enzyme can be selected from DHA kinase, methanol oxidase,PQQ-dependent methanol dehydrogenase, DHA synthase or any combinationthereof. Accordingly, in some aspects, the attenuation is of theendogenous enzyme DHA kinase. In some aspects, the attenuation is of theendogenous enzyme methanol oxidase. In some aspects, the attenuation isof the endogenous enzyme PQQ-dependent methanol dehydrogenase. In someaspects, the attenuation is of the endogenous enzyme DHA synthase. Theinvention also provides a microbial organism wherein attenuation is ofany combination of two or three endogenous enzymes described herein. Forexample, a microbial organism of the invention can include attenuationof DHA kinase and DHA synthase, or alternatively methanol oxidase andPQQ-dependent methanol dehydrogenase, or alternatively DHA kinase,methanol oxidase, and PQQ-dependent methanol dehydrogenase, oralternatively DHA kinase, methanol oxidase, and DHA synthase. Theinvention also provides a microbial organism wherein attenuation is ofall endogenous enzymes described herein. For example, in some aspects, amicrobial organism described herein includes attenuation of DHA kinase,methanol oxidase, PQQ-dependent methanol dehydrogenase and DHA synthase.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism as described herein, wherein the microbial organismfurther includes attenuation of one or more endogenous enzymes of acompeting formaldehyde assimilation or dissimilation pathway. Examplesof these endogenous enzymes are disclosed in FIG. 1 and described inExample XIII. It is understood that a person skilled in the art would beable to readily identify enzymes of such competing pathways. Competingpathways can be dependent upon the host microbial organism and/or theexogenous nucleic acid introduced into the microbial organism asdescribed herein. Accordingly, in some aspects of the invention, themicrobial organism includes attenuation of one, two, three, four, five,six, seven, eight, nine, ten or more endogenous enzymes of a competingformaldehyde assimilation or dissimilation pathway.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism as described herein, wherein the microbial organismfurther includes a gene disruption of one or more endogenous nucleicacids encoding enzymes, which enhances carbon flux through acetyl-CoA.For example, in some aspects, the endogenous enzyme can be selected fromDHA kinase, methanol oxidase, PQQ-dependent methanol dehydrogenase, DHAsynthase or any combination thereof. According, in some aspects, thegene disruptiondisruption is of an endogenous nucleic acid encoding theenzyme DHA kinase. In some aspects, the gene disruptiondisruption is ofan endogenous nucleic acid encoding the enzyme methanol oxidase. In someaspects, the gene disruptiondisruption is of an endogenous nucleic acidencoding the enzyme PQQ-dependent methanol dehydrogenase. In someaspects, the gene disruption is of an endogenous nucleic acid encodingthe enzyme DHA synthase. The invention also provides a microbialorganism wherein the gene disruption is of any combination of two orthree nucleic acids encoding endogenous enzymes described herein. Forexample, a microbial organism of the invention can include a genedisruption of DHA kinase and DHA synthase, or alternatively methanoloxidase and PQQ-dependent methanol dehydrogenase, or alternatively DHAkinase, methanol oxidase, and PQQ-dependent methanol dehydrogenase, oralternatively DHA kinase, methanol oxidase, and DHA synthase. Theinvention also provides a microbial organism wherein all endogenousnucleic acids encoding enzymes described herein are disrupted. Forexample, in some aspects, a microbial organism described herein includesdisruption of DHA kinase, methanol oxidase, PQQ-dependent methanoldehydrogenase and DHA synthase.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism as described herein, wherein the microbial organismfurther includes a gene disruption of one or more endogenous enzymes ofa competing formaldehyde assimilation or dissimilation pathway. Examplesof these endogenous enzymes are disclosed in FIG. 1 and described inExample XIII. It is understood that a person skilled in the art would beable to readily identify enzymes of such competing pathways. Competingpathways can be dependent upon the host microbial organism and/or theexogenous nucleic acid introduced into the microbial organism asdescribed herein. Accordingly, in some aspects of the invention, themicrobial organism includes a gene disruption of one, two, three, four,five, six, seven, eight, nine, ten or more endogenous nucleic acidsencoding enzymes of a competing formaldehyde assimilation ordissimilation pathway.

In the case of gene disruptions, a particularly useful stable geneticalteration is a gene deletion. The use of a gene deletion to introduce astable genetic alteration is particularly useful to reduce thelikelihood of a reversion to a phenotype prior to the geneticalteration. For example, stable growth-coupled production of abiochemical can be achieved, for example, by deletion of a gene encodingan enzyme catalyzing one or more reactions within a set of metabolicmodifications. The stability of growth-coupled production of abiochemical can be further enhanced through multiple deletions,significantly reducing the likelihood of multiple compensatoryreversions occurring for each disrupted activity.

Also provided is a method of producing a non-naturally occurringmicrobial organisms having stable growth-coupled production ofacetyl-CoA or a bioderived compound. The method can include identifyingin silico a set of metabolic modifications that increase production ofacetyl-CoA or a bioderived compound, for example, increase productionduring exponential growth; genetically modifying an organism to containthe set of metabolic modifications that increase production ofacetyl-CoA or a bioderived compound, and culturing the geneticallymodified organism. If desired, culturing can include adaptively evolvingthe genetically modified organism under conditions requiring productionof acetyl-CoA or a bioderived compound. The methods of the invention areapplicable to bacterium, yeast and fungus as well as a variety of othercells and microorganism, as disclosed herein.

Thus, the invention provides a non-naturally occurring microbialorganism comprising one or more gene disruptions that confer increasedsynthesis or production of acetyl-CoA or a bioderived compound. In oneembodiment, the one or more gene disruptions confer growth-coupledproduction of acetyl-CoA or a bioderived compound, and can, for example,confer stable growth-coupled production of acetyl-CoA or a bioderivedcompound. In another embodiment, the one or more gene disruptions canconfer obligatory coupling of acetyl-CoA or a bioderived compoundproduction to growth of the microbial organism. Such one or more genedisruptions reduce the activity of the respective one or more encodedenzymes.

The non-naturally occurring microbial organism can have one or more genedisruptions included in a gene encoding a enzyme or protein disclosedherein. As disclosed herein, the one or more gene disruptions can be adeletion. Such non-naturally occurring microbial organisms of theinvention include bacteria, yeast, fungus, or any of a variety of othermicroorganisms applicable to fermentation processes, as disclosedherein.

Thus, the invention provides a non-naturally occurring microbialorganism, comprising one or more gene disruptions, where the one or moregene disruptions occur in genes encoding proteins or enzymes where theone or more gene disruptions confer increased production of acetyl-CoAor a bioderived compound. The production of acetyl-CoA or a bioderivedcompound can be growth-coupled or not growth-coupled. In a particularembodiment, the production of acetyl-CoA or a bioderived compound can beobligatorily coupled to growth of the organism, as disclosed herein.

The invention provides non naturally occurring microbial organismshaving genetic alterations such as gene disruptions that increaseproduction of acetyl-CoA or a bioderived compound, for example,growth-coupled production of acetyl-CoA or a bioderived compound.Product production can be, for example, obligatorily linked to theexponential growth phase of the microorganism by genetically alteringthe metabolic pathways of the cell, as disclosed herein. The geneticalterations can increase the production of the desired product or evenmake the desired product an obligatory product during the growth phase.Metabolic alterations or transformations that result in increasedproduction and elevated levels of acetyl-CoA or a bioderived compoundbiosynthesis are exemplified herein. Each alteration corresponds to therequisite metabolic reaction that should be functionally disrupted.Functional disruption of all reactions within one or more of thepathways can result in the increased production of acetyl-CoA or abioderived compound by the engineered strain during the growth phase.

Each of these non-naturally occurring alterations result in increasedproduction and an enhanced level of acetyl-CoA or a bioderived compound,for example, during the exponential growth phase of the microbialorganism, compared to a strain that does not contain such metabolicalterations, under appropriate culture conditions. Appropriateconditions include, for example, those disclosed herein, includingconditions such as particular carbon sources or reactant availabilitiesand/or adaptive evolution.

Given the teachings and guidance provided herein, those skilled in theart will understand that to introduce a metabolic alteration such asattenuation of an enzyme, it can be necessary to disrupt the catalyticactivity of the one or more enzymes involved in the reaction.Alternatively, a metabolic alteration can include disrupting expressionof a regulatory protein or cofactor necessary for enzyme activity ormaximal activity. Furthermore, genetic loss of a cofactor necessary foran enzymatic reaction can also have the same effect as a disruption ofthe gene encoding the enzyme. Disruption can occur by a variety ofmethods including, for example, deletion of an encoding gene orincorporation of a genetic alteration in one or more of the encodinggene sequences. The encoding genes targeted for disruption can be one,some, or all of the genes encoding enzymes involved in the catalyticactivity. For example, where a single enzyme is involved in a targetedcatalytic activity, disruption can occur by a genetic alteration thatreduces or eliminates the catalytic activity of the encoded geneproduct. Similarly, where the single enzyme is multimeric, includingheteromeric, disruption can occur by a genetic alteration that reducesor destroys the function of one or all subunits of the encoded geneproducts. Destruction of activity can be accomplished by loss of thebinding activity of one or more subunits required to form an activecomplex, by destruction of the catalytic subunit of the multimericcomplex or by both. Other functions of multimeric protein associationand activity also can be targeted in order to disrupt a metabolicreaction of the invention. Such other functions are well known to thoseskilled in the art. Similarly, a target enzyme activity can be reducedor eliminated by disrupting expression of a protein or enzyme thatmodifies and/or activates the target enzyme, for example, a moleculerequired to convert an apoenzyme to a holoenzyme. Further, some or allof the functions of a single polypeptide or multimeric complex can bedisrupted according to the invention in order to reduce or abolish thecatalytic activity of one or more enzymes involved in a reaction ormetabolic modification of the invention. Similarly, some or all ofenzymes involved in a reaction or metabolic modification of theinvention can be disrupted so long as the targeted reaction is reducedor eliminated.

Given the teachings and guidance provided herein, those skilled in theart also will understand that an enzymatic reaction can be disrupted byreducing or eliminating reactions encoded by a common gene and/or by oneor more orthologs of that gene exhibiting similar or substantially thesame activity. Reduction of both the common gene and all orthologs canlead to complete abolishment of any catalytic activity of a targetedreaction. However, disruption of either the common gene or one or moreorthologs can lead to a reduction in the catalytic activity of thetargeted reaction sufficient to promote coupling of growth to productbiosynthesis. Exemplified herein are both the common genes encodingcatalytic activities for a variety of metabolic modifications as well astheir orthologs. Those skilled in the art will understand thatdisruption of some or all of the genes encoding a enzyme of a targetedmetabolic reaction can be practiced in the methods of the invention andincorporated into the non-naturally occurring microbial organisms of theinvention in order to achieve the increased production of acetyl-CoA ora bioderived compound or growth-coupled product production.

Given the teachings and guidance provided herein, those skilled in theart also will understand that enzymatic activity or expression can beattenuated using well known methods. Reduction of the activity or amountof an enzyme can mimic complete disruption of a gene if the reductioncauses activity of the enzyme to fall below a critical level that isnormally required for a pathway to function. Reduction of enzymaticactivity by various techniques rather than use of a gene disruption canbe important for an organism's viability. Methods of reducing enzymaticactivity that result in similar or identical effects of a genedisruption include, but are not limited to: reducing gene transcriptionor translation; destabilizing mRNA, protein or catalytic RNA; andmutating a gene that affects enzyme activity or kinetics (See, Sambrooket al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold SpringHarbor Laboratory, New York (2001); and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1999). Natural or imposed regulatory controls can also accomplishenzyme attenuation including: promoter replacement (See, Wang et al.,Mol. Biotechnol. 52(2):300-308 (2012)); loss or alteration oftranscription factors (Dietrick et al., Annu. Rev. Biochem. 79:563-590(2010); and Simicevic et al., Mol. Biosyst. 6(3):462-468 (2010));introduction of inhibitory RNAs or peptides such as siRNA, antisenseRNA, RNA or peptide/small-molecule binding aptamers, ribozymes,aptazymes and riboswitches (Wieland et al., Methods 56(3):351-357(2012); O'Sullivan, Anal. Bioanal. Chem. 372(1):44-48 (2002); and Lee etal., Curr. Opin. Biotechnol. 14(5):505-511(2003)); and addition of drugsor other chemicals that reduce or disrupt enzymatic activity such as anenzyme inhibitor, an antibiotic or a target-specific drug.

One skilled in the art will also understand and recognize thatattenuation of an enzyme can be done at various levels. For example, atthe gene level, a mutation causing a partial or complete null phenotype,such as a gene disruption, or a mutation causing epistatic geneticeffects that mask the activity of a gene product (Miko, Nature Education1(1) (2008)), can be used to attenuate an enzyme. At the gene expressionlevel, methods for attenuation include: coupling transcription to anendogenous or exogenous inducer, such as isopropylthio-β-galactoside(IPTG), then adding low amounts of inducer or no inducer during theproduction phase (Donovan et al., J. Ind. Microbiol. 16(3):145-154(1996); and Hansen et al., Curr. Microbiol. 36(6):341-347 (1998));introducing or modifying a positive or a negative regulator of a gene;modify histone acetylation/deacetylation in a eukaryotic chromosomalregion where a gene is integrated (Yang et al., Curr. Opin. Genet. Dev.13(2):143-153 (2003) and Kurdistani et al., Nat. Rev. Mol. Cell Biol.4(4):276-284 (2003)); introducing a transposition to disrupt a promoteror a regulatory gene (Bleykasten-Brosshans et al., C. R Biol.33(8-9):679-686 (2011); and McCue et al., PLoS Genet. 8(2):e1002474(2012)); flipping the orientation of a transposable element or promoterregion so as to modulate gene expression of an adjacent gene (Wang etal., Genetics 120(4):875-885 (1988); Hayes, Annu. Rev. Genet. 37:3-29(2003); in a diploid organism, deleting one allele resulting in loss ofheterozygosity (Daigaku et al., Mutation Research/Fundamental andMolecular Mechanisms of Mutagenesis 600(1-2)177-183 (2006)); introducingnucleic acids that increase RNA degradation (Houseley et al., Cell,136(4):763-776 (2009); or in bacteria, for example, introduction of atransfer-messenger RNA (tmRNA) tag, which can lead to RNA degradationand ribosomal stalling (Sunoham et al., RNA 10(3):378-386 (2004); andSunoham et al., J. Biol. Chem. 279:15368-15375 (2004)). At thetranslational level, attenuation can include: introducing rare codons tolimit translation (Angov, Biotechnol. J. 6(6):650-659 (2011));introducing RNA interference molecules that block translation (Castel etal., Nat. Rev. Genet. 14(2):100-112 (2013); and Kawasaki et al., Curr.Opin. Mol. Ther. 7(2):125-131(2005); modifying regions outside thecoding sequence, such as introducing secondary structure into anuntranslated region (UTR) to block translation or reduce efficiency oftranslation (Ringnér et al., PLoS Comput. Biol. 1(7):e72 (2005)); addingRNAase sites for rapid transcript degradation (Pasquinelli, Nat. Rev.Genet. 13(4):271-282 (2012); and Anaiano et al., FEMS Microbiol. Rev.34(5):883-932 (2010); introducing antisense RNA oligomers or antisensetranscripts (Nashizawa et al., Front. Biosci. 17:938-958 (2012));introducing RNA or peptide aptamers, ribozymes, aptazymes, riboswitches(Wieland et al., Methods 56(3):351-357 (2012); O'Sullivan, Anal.Bioanal. Chem. 372(1):44-48 (2002); and Lee et al., Curr. Opin.Biotechnol. 14(5):505-511(2003)); or introducing translationalregulatory elements involving RNA structure that can prevent or reducetranslation that can be controlled by the presence or absence of smallmolecules (Araujo et al., Comparative and Functional Genomics, ArticleID 475731, 8 pages (2012)). At the level of enzyme localization and/orlongevity, enzyme attenuation can include: adding a degradation tag forfaster protein turnover (Hochstrasser, Annual Rev. Genet. 30:405-439(1996); and Yuan et al., PLoS One 8(4):e62529 (2013)); or adding alocalization tag that results in the enzyme being secreted or localizedto a subcellular compartment in a eukaryotic cell, where the enzymewould not be able to react with its normal substrate (Nakai et al.Genomics 14(4):897-911 (1992); and Russell et al., J. Bact.189(21)7581-7585 (2007)). At the level of post-translational regulation,enzyme attenuation can include: increasing intracellular concentrationof known inhibitors; or modifying post-translational modified sites(Mann et al., Nature Biotech. 21:255-261 (2003)). At the level of enzymeactivity, enzyme attenuation can include: adding an endogenous or anexogenous inhibitor, such as an enzyme inhibitor, an antibiotic or atarget-specific drug, to reduce enzyme activity; limiting availabilityof essential cofactors, such as vitamin B12, for an enzyme that requiresthe cofactor; chelating a metal ion that is required for enzymeactivity; or introducing a dominant negative mutation. The applicabilityof a technique for attenuation described above can depend upon whether agiven host microbial organism is prokaryotic or eukaryotic, and it isunderstand that a determination of what is the appropriate technique fora given host can be readily made by one skilled in the art.

In some embodiments, microaerobic designs can be used based on thegrowth-coupled formation of the desired product. To examine this,production cones can be constructed for each strategy by firstmaximizing and, subsequently minimizing the product yields at differentrates of biomass formation feasible in the network. If the rightmostboundary of all possible phenotypes of the mutant network is a singlepoint, it implies that there is a unique optimum yield of the product atthe maximum biomass formation rate possible in the network. In othercases, the rightmost boundary of the feasible phenotypes is a verticalline, indicating that at the point of maximum biomass the network canmake any amount of the product in the calculated range, including thelowest amount at the bottommost point of the vertical line. Such designsare given a low priority.

The acetyl-CoA or a bioderived compound production strategies identifiedherein can be disrupted to increase production of acetyl-CoA or abioderived compound. Accordingly, the invention also provides anon-naturally occurring microbial organism having metabolicmodifications coupling acetyl-CoA or a bioderived compound production togrowth of the organism, where the metabolic modifications includesdisruption of one or more genes selected from the genes encodingproteins and/or enzymes shown in the various tables disclosed herein.

Each of the strains can be supplemented with additional deletions if itis determined that the strain designs do not sufficiently increase theproduction of acetyl-CoA or a bioderived compound and/or couple theformation of the product with biomass formation. Alternatively, someother enzymes not known to possess significant activity under the growthconditions can become active due to adaptive evolution or randommutagenesis. Such activities can also be knocked out. However, the genesdisclosed herein allows the construction of strains exhibitinghigh-yield synthesis or production of acetyl-CoA or a bioderivedcompound, including growth-coupled production of acetyl-CoA or abioderived compound.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art will understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reaction,and reference to any of these metabolic constituents also references thegene or genes encoding the enzymes that catalyze or proteins involved inthe referenced reaction, reactant or product. Likewise, given the wellknown fields of metabolic biochemistry, enzymology and genomics,reference herein to a gene or encoding nucleic acid also constitutes areference to the corresponding encoded enzyme and the reaction itcatalyzes or a protein associated with the reaction as well as thereactants and products of the reaction.

As disclosed herein, the bioderived compounds adipate, 6-aminocaproate,methacrylic acid, 2-hydroxyisobutyric acid, as well as otherintermediates, are carboxylic acids, which can occur in various ionizedforms, including fully protonated, partially protonated, and fullydeprotonated forms. Accordingly, the suffix “-ate,” or the acid form,can be used interchangeably to describe both the free acid form as wellas any deprotonated form, in particular since the ionized form is knownto depend on the pH in which the compound is found. It is understoodthat carboxylate products or intermediates includes ester forms ofcarboxylate products or pathway intermediates, such as O-carboxylate andS-carboxylate esters. O- and S-carboxylates can include lower alkyl,that is C1 to C6, branched or straight chain carboxylates. Some such O-or S-carboxylates include, without limitation, methyl, ethyl, n-propyl,n-butyl, i-propyl, sec-butyl, and tert-butyl, pentyl, hexyl O- orS-carboxylates, any of which can further possess an unsaturation,providing for example, propenyl, butenyl, pentyl, and hexenyl O- orS-carboxylates. O-carboxylates can be the product of a biosyntheticpathway. Exemplary O-carboxylates accessed via biosynthetic pathways caninclude, without limitation, methyl adipate, ethyl adipate, and n-propyladipate. Other biosynthetically accessible O-carboxylates can includemedium to long chain groups, that is C7-C22, O-carboxylate estersderived from fatty alcohols, such heptyl, octyl, nonyl, decyl, undecyl,lauryl, tridecyl, myristyl, pentadecyl, cetyl, palmitolyl, heptadecyl,stearyl, nonadecyl, arachidyl, heneicosyl, and behenyl alcohols, any oneof which can be optionally branched and/or contain unsaturations.O-carboxylate esters can also be accessed via a biochemical or chemicalprocess, such as esterification of a free carboxylic acid product ortransesterification of an O- or S-carboxylate. S-carboxylates areexemplified by CoA S-esters, cysteinyl S-esters, alkylthioesters, andvarious aryl and heteroaryl thioesters.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes or proteins participating in one or more acetyl-CoA or abioderived compound biosynthetic pathways. Depending on the hostmicrobial organism chosen for biosynthesis, nucleic acids for some orall of a particular acetyl-CoA or a bioderived compound biosyntheticpathway can be expressed. For example, if a chosen host is deficient inone or more enzymes or proteins for a desired biosynthetic pathway, thenexpressible nucleic acids for the deficient enzyme(s) or protein(s) areintroduced into the host for subsequent exogenous expression.Alternatively, if the chosen host exhibits endogenous expression of somepathway genes, but is deficient in others, then an encoding nucleic acidis needed for the deficient enzyme(s) or protein(s) to achieveacetyl-CoA or a bioderived compound biosynthesis. Thus, a non-naturallyoccurring microbial organism of the invention can be produced byintroducing exogenous enzyme or protein activities to obtain a desiredbiosynthetic pathway or a desired biosynthetic pathway can be obtainedby introducing one or more exogenous enzyme or protein activities that,together with one or more endogenous enzymes or proteins, produces adesired product such as acetyl-CoA or a bioderived compound.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable orsuitable to fermentation processes. Exemplary bacteria include anyspecies selected from the order Enterobacteriales, familyEnterobacteriaceae, including the genera Escherichia and Klebsiella, theorder Aeromonadales, family Succinivibrionaceae, including the genusAnaerobiospirillum; the order Pasteurellales, family Pasteurellaceae,including the genera Actinobacillus and Mannheimia; the orderRhizobiales, family Bradyrhizobiaceae, including the genus Rhizobium;the order Bacillales, family Bacillaceae, including the genus Bacillus;the order Actinomycetales, families Corynebacteriaceae andStreptomycetaceae, including the genus Corynebacterium and the genusStreptomyces, respectively; order Rhodospirillales, familyAcetobacteraceae, including the genus Gluconobacter; the orderSphingomonadales, family Sphingomonadaceae, including the genusZymomonas; the order Lactobacillales, families Lactobacillaceae andStreptococcaceae, including the genus Lactobacillus and the genusLactococcus, respectively; the order Clostridiales, familyClostridiaceae, genus Clostridium; and the order Pseudomonadales, familyPseudomonadaceae, including the genus Pseudomonas. Non-limiting speciesof host bacteria include Escherichia coli, Klebsiella oxytoca,Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes,Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis,Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis,Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor,Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonasputida. Exemplary bacterial methylotrophs include, for example,Bacillus, Methylobacterium, Methyloversatilis, Methylococcus,Methylocystis and Hyphomicrobium.

Similarly, exemplary species of yeast or fungi species include anyspecies selected from the order Saccharomycetales, familySaccaromycetaceae, including the genera Saccharomyces, Kluyveromyces andPichia; the order Saccharomycetales, family Dipodascaceae, including thegenus Yarrowia; the order Schizosaccharomycetales, familySchizosaccaromycetaceae, including the genus Schizosaccharomyces; theorder Eurotiales, family Trichocomaceae, including the genusAspergillus; and the order Mucorales, family Mucoraceae, including thegenus Rhizopus. Non-limiting species of host yeast or fungi includeSaccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyceslactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger,Pichia pastor's, Rhizopus arrhizus, Rhizopus oryzae, Yarrowialipolytica, and the like. E. coli is a particularly useful host organismsince it is a well characterized microbial organism suitable for geneticengineering. Other particularly useful host organisms include yeast suchas Saccharomyces cerevisiae and yeasts or fungi selected from the generaSaccharomyces, Schizosaccharomyces, Schizochytrium, Rhodotorula,Thraustochytrium, Aspergillus, Kluyveromyces, Issatchenkia, Yarrowia,Candida, Pichia, Ogataea, Kuraishia, Hansenula and Komagataella. Usefulhost organisms include Saccharomyces cerevisiae, Schizosaccharomycespombe, Hansenula polymorpha, Pichia methanolica, Candida boidinii,Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus,Aspergillus niger, Pichia pastor's, Rhizopus arrhizus, Rhizopus oryzae,Yarrowia lipolytica, Issatchenkia orientalis and the like. It isunderstood that any suitable microbial host organism can be used tointroduce metabolic and/or genetic modifications to produce a desiredproduct.

Depending on the acetyl-CoA or the bioderived compound biosyntheticpathway constituents of a selected host microbial organism, thenon-naturally occurring microbial organisms of the invention willinclude at least one exogenously expressed acetyl-CoA or a bioderivedcompound pathway-encoding nucleic acid and up to all encoding nucleicacids for one or more acetyl-CoA or a bioderived compound biosyntheticpathways. For example, acetyl-CoA or a bioderived compound biosynthesiscan be established in a host deficient in a pathway enzyme or proteinthrough exogenous expression of the corresponding encoding nucleic acid.In a host deficient in all enzymes or proteins of an acetyl-CoA or abioderived compound pathway, exogenous expression of all enzyme orproteins in the pathway can be included, although it is understood thatall enzymes or proteins of a pathway can be expressed even if the hostcontains at least one of the pathway enzymes or proteins. For example,exogenous expression of all enzymes or proteins in a pathway forproduction of acetyl-CoA or a bioderived compound can be included, suchas a fructose-6-phosphate phosphoketolase and a phosphotransacetylase(see, e.g. FIG. 1), or a xylulose-5-phosphate phosphoketolase and aphosphotransacetylase (see, e.g. FIG. 1), or a methanol dehydrogenase, a3-hexulose-6-phosphate synthase, a 6-phospho-3-hexuloisomerase, afructose-6-phosphate phosphoketolase and a phosphotransacetylase (see,e.g. FIG. 1), or an acetyl-CoA:acetyl-CoA acyltransferase, anacetoacetyl-CoA reductase (ketone reducing), a 3-hydroxybutyryl-CoAreductase (aldehyde forming), and a 3-hydroxybutyraldehyde reductase(see, e.g. FIG. 5), or a succinyl-CoA reductase (aldehyde forming), a4-HB dehydrogenase, a 4-HB kinase, a phosphotrans-4-hydroxybutyrylase, a4-hydroxybutyryl-CoA reductase (aldehyde forming), and a 1,4-butanedioldehydrogenase (see, e.g. FIG. 7), or a 3-oxoadipyl-CoA thiolase, a3-oxoadipyl-CoA reductase, a 3-hydroxyadipyl-CoA dehydratase, a5-carboxy-2-pentenoyl-CoA reductase, an adipyl-CoA reductase (aldehydeforming), and 6-aminocaproate transaminase (see, e.g. FIG. 8), or anacetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase(ketone reducing), a 3-hydroxybutyrl-CoA mutase, a2-hydroxyisobutyryl-CoA dehydratase, and a methacrylyl-CoA synthetase(see, e.g. FIG. 10).

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel the acetyl-CoAor the bioderived compound pathway deficiencies of the selected hostmicrobial organism. Therefore, a non-naturally occurring microbialorganism of the invention can have one, two, three, four, five, six,seven, eight, nine, ten, eleven, or twelve up to all nucleic acidsencoding the enzymes or proteins constituting an acetyl-CoA or abioderived compound biosynthetic pathway disclosed herein. In someembodiments, the non-naturally occurring microbial organisms also caninclude other genetic modifications that facilitate or optimizeacetyl-CoA or a bioderived compound biosynthesis or that confer otheruseful functions onto the host microbial organism. One such otherfunctionality can include, for example, augmentation of the synthesis ofone or more of the acetyl-CoA or the bioderived compound pathwayprecursors such as Fald, H6P, DHA, G3P, malonyl-CoA, acetoacetyl-CoA,PEP, PYR and Succinyl-CoA.

Generally, a host microbial organism is selected such that it producesthe precursor of an acetyl-CoA or a bioderived compound pathway, eitheras a naturally produced molecule or as an engineered product that eitherprovides de novo production of a desired precursor or increasedproduction of a precursor naturally produced by the host microbialorganism. For example, malonyl-CoA, acetoacetyl-CoA and pyruvate areproduced naturally in a host organism such as E. coli. A host organismcan be engineered to increase production of a precursor, as disclosedherein. In addition, a microbial organism that has been engineered toproduce a desired precursor can be used as a host organism and furtherengineered to express enzymes or proteins of an acetyl-CoA or abioderived compound pathway.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize acetyl-CoA or a bioderived compound. In thisspecific embodiment it can be useful to increase the synthesis oraccumulation of an acetyl-CoA or a bioderived compound pathway productto, for example, drive acetyl-CoA or a bioderived compound pathwayreactions toward acetyl-CoA or a bioderived compound production.Increased synthesis or accumulation can be accomplished by, for example,overexpression of nucleic acids encoding one or more of theabove-described acetyl-CoA or a bioderived compound pathway enzymes orproteins. Overexpression of the enzyme or enzymes and/or protein orproteins of the acetyl-CoA or the bioderived compound pathway can occur,for example, through exogenous expression of the endogenous gene orgenes, or through exogenous expression of the heterologous gene orgenes. Therefore, naturally occurring organisms can be readily generatedto be non-naturally occurring microbial organisms of the invention, forexample, producing acetyl-CoA or a bioderived compound, throughoverexpression of one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, that is, up to all nucleic acids encodingacetyl-CoA or a bioderived compound biosynthetic pathway enzymes orproteins. In addition, a non-naturally occurring organism can begenerated by mutagenesis of an endogenous gene that results in anincrease in activity of an enzyme in the acetyl-CoA or the bioderivedcompound biosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, an acetyl-CoA or a bioderived compound biosynthetic pathwayonto the microbial organism. Alternatively, encoding nucleic acids canbe introduced to produce an intermediate microbial organism having thebiosynthetic capability to catalyze some of the required reactions toconfer acetyl-CoA or a bioderived compound biosynthetic capability. Forexample, a non-naturally occurring microbial organism having anacetyl-CoA or a bioderived compound biosynthetic pathway can comprise atleast two exogenous nucleic acids encoding desired enzymes or proteins,such as the combination of a 3-hexulose-6-phosphate synthase and afructose-6-phosphate phosphoketolase, or alternatively axylulose-5-phosphate phosphoketolase and an acetyl-CoA transferase, oralternatively a fructose-6-phosphate phosphoketolase and a formatereductase, or alternatively a xylulose-5-phosphate phosphoketolase and amethanol dehydrogenase, and the like. Thus, it is understood that anycombination of two or more enzymes or proteins of a biosynthetic pathwaycan be included in a non-naturally occurring microbial organism of theinvention. Similarly, it is understood that any combination of three ormore enzymes or proteins of a biosynthetic pathway can be included in anon-naturally occurring microbial organism of the invention, forexample, a methanol dehydrogenase, a fructose-6-phosphate aldolase, anda fructose-6-phosphate phosphoketolase, or alternatively a methanolmethyltransferase, fructose-6-phosphate phosphoketolase and a3-hydroxybutyraldehyde reductase, or alternatively axylulose-5-phosphate phosphoketolase, a pyruvate formate lyase and a4-hydroxybutyryl-CoA reductase (alcohol forming), or alternatively afructose-6-phosphate aldolase, a phosphotransacetylase, and a3-hydroxyisobutyrate dehydratase, and so forth, as desired, so long asthe combination of enzymes and/or proteins of the desired biosyntheticpathway results in production of the corresponding desired product.Similarly, any combination of four, five, six, seven, eight, nine, ten,eleven, twelve or more enzymes or proteins of a biosynthetic pathway asdisclosed herein can be included in a non-naturally occurring microbialorganism of the invention, as desired, so long as the combination ofenzymes and/or proteins of the desired biosynthetic pathway results inproduction of the corresponding desired product.

In addition to the biosynthesis of acetyl-CoA or a bioderived compoundas described herein, the non-naturally occurring microbial organisms andmethods of the invention also can be utilized in various combinationswith each other and/or with other microbial organisms and methods wellknown in the art to achieve product biosynthesis by other routes. Forexample, one alternative to produce acetyl-CoA or a bioderived compoundother than use of the acetyl-CoA or the bioderived compound producers isthrough addition of another microbial organism capable of converting anacetyl-CoA or a bioderived compound pathway intermediate to acetyl-CoAor a bioderived compound. One such procedure includes, for example, thefermentation of a microbial organism that produces an acetyl-CoA or abioderived compound pathway intermediate. The acetyl-CoA or thebioderived compound pathway intermediate can then be used as a substratefor a second microbial organism that converts the acetyl-CoA or thebioderived compound pathway intermediate to acetyl-CoA or a bioderivedcompound. The acetyl-CoA or the bioderived compound pathway intermediatecan be added directly to another culture of the second organism or theoriginal culture of the acetyl-CoA or the bioderived compound pathwayintermediate producers can be depleted of these microbial organisms by,for example, cell separation, and then subsequent addition of the secondorganism to the fermentation broth can be utilized to produce the finalproduct without intermediate purification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, acetyl-CoA or abioderived compound. In these embodiments, biosynthetic pathways for adesired product of the invention can be segregated into differentmicrobial organisms, and the different microbial organisms can beco-cultured to produce the final product. In such a biosynthetic scheme,the product of one microbial organism is the substrate for a secondmicrobial organism until the final product is synthesized. For example,the biosynthesis of acetyl-CoA or a bioderived compound can beaccomplished by constructing a microbial organism that containsbiosynthetic pathways for conversion of one pathway intermediate toanother pathway intermediate or the product. Alternatively, acetyl-CoAor a bioderived compound also can be biosynthetically produced frommicrobial organisms through co-culture or co-fermentation using twoorganisms in the same vessel, where the first microbial organismproduces an acetyl-CoA or a bioderived compound intermediate and thesecond microbial organism converts the intermediate to acetyl-CoA or abioderived compound.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce acetyl-CoA or a bioderivedcompound.

Similarly, it is understood by those skilled in the art that a hostorganism can be selected based on desired characteristics forintroduction of one or more gene disruptions to increase synthesis orproduction of acetyl-CoA or a bioderived compound. Thus, it isunderstood that, if a genetic modification is to be introduced into ahost organism to disrupt a gene, any homologs, orthologs or paralogsthat catalyze similar, yet non-identical metabolic reactions cansimilarly be disrupted to ensure that a desired metabolic reaction issufficiently disrupted. Because certain differences exist amongmetabolic networks between different organisms, those skilled in the artwill understand that the actual genes disrupted in a given organism maydiffer between organisms. However, given the teachings and guidanceprovided herein, those skilled in the art also will understand that themethods of the invention can be applied to any suitable hostmicroorganism to identify the cognate metabolic alterations needed toconstruct an organism in a species of interest that will increaseacetyl-CoA or a bioderived compound biosynthesis. In a particularembodiment, the increased production couples biosynthesis of acetyl-CoAor a bioderived compound to growth of the organism, and can obligatorilycouple production of acetyl-CoA or a bioderived compound to growth ofthe organism if desired and as disclosed herein.

Sources of encoding nucleic acids for an acetyl-CoA or a bioderivedcompound pathway enzyme or protein can include, for example, any specieswhere the encoded gene product is capable of catalyzing the referencedreaction. Such species include both prokaryotic and eukaryotic organismsincluding, but not limited to, bacteria, including archaea andeubacteria, and eukaryotes, including yeast, plant, insect, animal, andmammal, including human. Exemplary species for such sources include, forexample, Escherichia coli, Abies grandis, Acetobacter aceti, Acetobacterpasteurians, Achromobacter denitnficans, Acidaminococcus fermentans,Acinetobacter baumannii Naval-82, Acinetobacter baylyi, Acinetobactercalcoaceticus, Acinetobacter sp. ADP1, Acinetobacter sp. Strain M-1,Actinobacillus succinogenes, Actinobacillus succinogenes 130Z, Aeropyrumpernix, Agrobacterium tumefaciens, Alkaliphilus metalliredigenes QYF,Allochromatium vinosum DSM 180, Aminomonas aminovorus, Amycolicicoccussubflavus DQS3-9A1, Anaerobiospirillum succiniciproducens, Anaerotruncuscolihominis, Aquifex aeolicus VF5, Arabidopsis thaliana, Arabidopsisthaliana col, Archaeglubus fulgidus, Archaeoglobus fulgidus,Archaeoglobus fulgidus DSM 4304, Arthrobacter globiformis, Ascaris suum,Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger,Aspergillus niger CBS 513.88, Aspergillus terreus MH2624, Atopobiumparvulum DSM 20469, Azotobacter vinelandii DJ, Bacillus alcalophilusATCC 27647, Bacillus azotoformans LMG 9581, Bacillus cereus, Bacilluscereus ATCC 14579, Bacillus coagulans 36D1, Bacillus megaterium,Bacillus methanolicus MGA3, Bacillus methanolicus PB1, Bacillusmethanolicus PB-1, Bacillus selenitireducens MLS10, Bacillus smithii,Bacillus sphaericus, Bacillus subtilis, Bacteroides capillosus,Bifidobacterium animalis lactis, Bifidobacterium breve, Bifidobacteriumdentium ATCC 27678, Bifidobacterium pseudolongum subsp. globosum, Bostaurus, Burkholderia cenocepacia, Burkholderia cepacia, Burkholderiamultivorans, Burkholderia pyrrocinia, Burkholderia stabilis,Burkholderia thailandensis E264, Burkholderia xenovorans, butyrateproducing bacterium L2-50, Caenorhabditis elegans, Campylobacter curvus525.92, Campylobacter jejuni, Candida albicans, Candida boidinii,Candida methylica, Candida parapsilosis, Candida tropicalis, Candidatropicalis MYA-3404, Carboxydothermus hydrogenoformans, Carboxydothermushydrogenoformans Z-2901, Castellaniella defragrans, Caulobacter sp.AP07, Chlamydomonas reinhardiii, Chlorobium phaeobacteroides DSM 266,Chlorobium limicola, Chlorobium tepidum, Chlorojlexus aggregans DSM9485, Chlorojlexus aurantiacus, Chlorojlexus aurantiacus J-10-fl,Citrobacter koseri ATCC BAA-895, Citrobacter youngae, Citrobacteryoungae ATCC 29220, Clostridium acetobutylicum, Clostridiumacetobutylicum ATCC 824, Clostridium acidurici, Clostridiumaminobutyricum, Clostridium asparagiforme DSM 15981, Clostridiumbeijerinckii, Clostridium beijerinckii NCIMB 8052, Clostridiumbeijerinckii NRRL B593, Clostridium beijerinckii, Clostridium bolteaeATCC BAA-613, Clostridium botulinum C str. Eklund, Clostridiumcarboxidivorans P7, Clostridium cellulolyticum H10, Clostridiumcellulovorans 743B, Clostridium difficile, Clostridium difficile 630,Clostridium hiranonis DSM 13275, Clostridium hylemonae DSM 15053,Clostridium kluyveri, Clostridium kluyveri DSM 555, Clostridiumljungdahli, Clostridium ljungdahlii DSM, Clostridium ljungdahlii DSM13528, Clostridium methylpentosum DSM 5476, Clostridium novyi NT,Clostridium pasteurianum, Clostridium pasteurianum DSM 525, Clostridiumperfringens, Clostridium perfringens ATCC 13124, Clostridium perfringensstr. 13, Clostridium phytofermentans ISDg, Clostridium propionicum,Clostridium saccharobutylicum, Clostridium saccharoperbutylacetonicum,Clostridium saccharoperbutylacetonicum N1-4, Clostridium tetani,Comamonas sp. CNB-1, Comamonas sp. CNB-1, Corynebacterium glutamicum,Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum R, Corynebacterium sp.,Corynebacterium sp. U-96, Corynebacterium variabile,Cryptosporidiumparvum Iowa II Cucumis sativus, Cupriavidus necatorN-1,Cyanobium PCC7001, Deinococcus radiodurans R1, Desulfatibacillumalkenivorans AK-01, Desulfotobacterium hafniense, Desulfotobacteriummetallireducens DSM15288, Desulfotomaculum reducens MI-1, Desulfovibrioafricanus, Desulfovibrio africanus str. Walvis Bay, DesulfoVibriodesulfuricans G20, Desulfovibrio desulfuricans subsp. desulfiuricanssfr. ATCC 27774, Desulfovibrio fructosovorans JJ, Desulfovibrio vulgarisstr. Hildenborough, Dictyostelium discoideum AX4, Elizabethkingiameningoseptica, Enterococcus faecalis, Erythrobacter sp. NAP1,Escherichia coli C, Escherichia coli K12, Escherichia coli K-12MG1655,Escherichia coli W, Eubacterium barkeri, Eubacterium hallii DSM3353,Eubacterium rectale ATCC 33656, Euglena gracilis, Flavobacteriumfrigoris, Fusobacterium nucleatum, Fusobacterium nucleatum subsp.polymorphum ATCC 10953, Geobacillus sp. GHH01, Geobacillus sp. M10EXG,Geobacillus sp. Y4.1MC1, Geobacillus stearothermophilus, Geobacillusthemodenitrijfcans NG80-2, Geobacillus thermoglucosidasius, Geobacterbemidjiensis Bem, Geobacter metallireducens GS-15, Geobactersulfurreducens, Geobacter suflfrreducens PCA, Haemophilus influenza,Haemophilus influenzae, Haloarcula marismortui, Haloarcula marismortuiATCC 43049, Halobacterium salinarum, Hansenula polymorpha DL-1,Helicobacter pylori, Helicobacter pylori 26695, Helicobacter pylori,Homo sapiens, human gut metagenome, Hydrogenobacter thermophilus,Hydrogenobacter thermophilus TK-6, Hyphomicrobium denitrifcans ATCC51888, Hyphomicrobium zavarzinii, Klebsiella pneumoniae, Klebsiellapneumoniae subsp. pneumoniae MGH 78578, Kluyveromyces lactis,Kluyveromyces lactis NRRL Y-1140, Lactobacillus acidophilus,Lactobacillus brevis ATCC 367, Lactobacillus paraplantarum, Lactococcuslactis, Leuconostoc mesenteroides, Lysinibacillus fusiformis,Lysinibacillus sphaericus, Malus x domestica, Mannheimiasucciniciproducens, marine gamma proteobacterium HTCC2080, Marinemetagenome JCVI SCAF 1096627185304, Mesorhizobium loti MAFF303099,Metallosphaera sedula, Metarhizium acridum CQMa 102, Methanocaldococcusjanaschii, Methanocaldococcus annaschii, Methanosarcina acetivorans,Methanosarcina acetivorans C2A, Methanosarcina barkeri, Methanosarcinamazei, Methanosarcina mazei Tuc01, Methanosarcina thermophila,Methanothermobacter thermautotrophicus, Methylibium petroleiphilum PM1,Methylobacillus flagellatus, Methylobacillus fagellatus KT Methylobactermarinus, Methylobacterium extorquens, Methylobacterium extorquens AM1,Methylococcus capsulatas, Methylomicrobium album BG8, Methylomonasaminofaciens, Methylovorus glucosetrophus SIP3-4, Methylovorus sp.MP688, Moorella thermoacetica, Mus musculus, Mycobacter sp. strain JC1DSM3803, Mycobacterium avium subsp. paratuberculosis K-10, Mycobacteriumbovis BCG, Mycobacterium gastri, Mycobacterium marinum M, Mycobacteriumsmegmatis, Mycobacterium smegmatis MC2 155, Mycobacterium tuberculosis,Mycoplasma pneumoniae M129, Natranaerobius thermophilus, Nectriahaematococca mpVI 77-13-4, Neurospora crassa, Nitrososphaera gargensisGa9.2, Nocardia brasiliensis, Nocardia farcinica IFM10152, Nocardiaiowensis, Nocardia iowensis (sp. NRRL 5646), Nostoc sp. PCC 7120,Ogataea parapolymorpha DL-1 (Hansenula polymorpha DL-1), Organism,Oryctolagus cuniculus, Oxalobacter formigenes, Paenibacillus peoriaeKCTC 3763, Paracoccus denitrificans, Pelobacter carbinolicus DSM2380,Pelotomaculum thermopropionicum, Penicillium chrysogenum, Perkinsusmarinus ATCC 50983, Photobacterium profundum 3TCK, Picea abies, Pichiapastoris, Picrophilus torridus DSM9790, Pinus sabiniana, Plasmodiumfalciparum, Populus alba, Populus tremula x Populus alba, Porphyromonasgingivalis, Porphyromonas gingivalis W83, Propionibacterium acnes,Propionibacterium fredenreichii sp. shermanii, Pseudomonas aeruginosa,Pseudomonas aemginosa PA01, Pseudomonas chlororaphis, Pseudomonasfluorescens, Pseudomonas knackmussii, Pseudomonas knackmussii (B13),Pseudomonas mendocina, Pseudomonas putida, Pseudomonas sp, Pseudomonassyringae pv. syringae B728a, Psychroflexus torquis ATCC 700755, Puerariamontana, Pyrobaculum aerophilum str. IM2, Pyrobaculum islandicumDSM4184, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshiiOT3, Ralstonia eutropha, Ralstonia eutropha H16, Rattus norvegicus,Rhizobium leguminosarum, Rhodobacter capsulatus, Rhodobactersphaeroides, Rhodobacter sphaeroides ATCC 17025, Rhodococcus opacus B4,Rhodococcus ruber, Rhodopseudomonas palustris, Rhodopseudomonaspalustris CGA009, Rhodospirillum rubrum, Rhodospirillum rubrum ATCC11170, Roseburia intestinalis L1-82, Roseburia inulinivorans, Roseburiasp. A2-183, Roseiflexus castenholzii, Rubrivivax gelatinosus,Ruminococcus obeum ATCC 29174, Saccharomyces cerevisiae, Saccharomycescerevisiae s288c, Saccharomyces kluyveri, Saccharomyces serevisiae,Sachharomyces cerevisiae, Salmonella enteric, Salmonella enterica,Salmonella enterica subsp. arizonae serovar, Salmonella enterica subsp.enterica serovar Typhimurium str. LT2, Salmonella enterica Typhimurium,Salmonella typhimurium, Salmonella typhimurium LT2, Schizosaccharomycespombe, Sebaldella termitidis ATCC 33386, Serratia proteamaculans,Shewanella oneidensis MR-1, Shigella flexneri, Sinorhizobium meliloti1021, Solanum lycopersicum, Staphylococcus aureus, Stereum hirsutumFP-91666 SS1, Streptococcus mutans, Streptococcus pneumonia,Streptococcus pneumoniae, Streptococcus pyogenes ATCC 10782,Streptomyces anulatus, Streptomyces avermitilis, Streptomycescinnamonensis, Streptomyces clavuligerus, Streptomyces coelicolor,Streptomyces coelicolor A3(2), Streptomyces griseus, Streptomycesgriseus subsp. griseus NBRC 13350, Streptomyces sp CL190, Streptomycessp. 2065, Streptomyces sp. ACT-1, Streptomyces sp. KO-3988, Sulfolobusacidocalarius, Sulfolobus acidocaldarius, Sulfolobus solfataricus,Sulfolobus solfataricus P-2, Sulfolobus sp. strain 7, Sulfolobustokodan, Sulfurimonas denitrifcans, Sus scrofa, Synechococcus elongatusPCC 7942, Synechococcus sp. PCC 7002, Synechocystis str. PCC 6803,Syntrophobacter fumaroxidans, Thauera aromatica, Thermoanaerobacterbrockii HTD4, Thermoanaerobacter sp. X514, Thermoanaerobactertengcongensis MB4, Thermococcus kodakaraensis, Thermococcus litoralis,Thermoplasma acidophilum, Thermoproteus neutrophilus, Thermotogamaritima, Thermotoga maritime, Thermotoga maritime MSB8, Thermusthermophilus, Thiocapsa roseopersicina, Tolumonas auensis DSM9187,Treponema denticola, Trichomonas vaginalis G3, Triticum aestivum,Trypanosoma brucei, Tsukamurella paurometabola DSM 20162, Unculturedbacterium, uncultured organism, Vibrio cholera, Vibrio harveyi ATCCBAA-1116, Xanthobacter autotrophicus Py2, Yarrowia lipolytica, Yersiniafrederiksenii, Yersinia intermedia, Yersinia intermedia ATCC 29909,Yersinia pestis, Zea mays, Zoogloea ramigera, Zymomonas mobilus, as wellas other exemplary species disclosed herein or available as sourceorganisms for corresponding genes. However, with the complete genomesequence available for now more than 550 species (with more than half ofthese available on public databases such as the NCBI), including 395microorganism genomes and a variety of yeast, fungi, plant, andmammalian genomes, the identification of genes encoding the requisiteacetyl-CoA or a bioderived compound biosynthetic activity for one ormore genes in related or distant species, including for example,homologues, orthologs, paralogs and nonorthologous gene displacements ofknown genes, and the interchange of genetic alterations betweenorganisms is routine and well known in the art. Accordingly, themetabolic alterations allowing biosynthesis of acetyl-CoA or abioderived compound described herein with reference to a particularorganism such as E. coli can be readily applied to other microorganisms,including prokaryotic and eukaryotic organisms alike. Given theteachings and guidance provided herein, those skilled in the art willknow that a metabolic alteration exemplified in one organism can beapplied equally to other organisms.

In some instances, such as when an alternative acetyl-CoA or abioderived compound biosynthetic pathway exists in an unrelated species,acetyl-CoA or a bioderived compound biosynthesis can be conferred ontothe host species by, for example, exogenous expression of a paralog orparalogs from the unrelated species that catalyzes a similar, yetnon-identical metabolic reaction to replace the referenced reaction.Because certain differences among metabolic networks exist betweendifferent organisms, those skilled in the art will understand that theactual gene usage between different organisms may differ. However, giventhe teachings and guidance provided herein, those skilled in the artalso will understand that the teachings and methods of the invention canbe applied to all microbial organisms using the cognate metabolicalterations to those exemplified herein to construct a microbialorganism in a species of interest that will synthesize acetyl-CoA or abioderived compound.

A nucleic acid molecule encoding an acetyl-CoA or a bioderived compoundpathway enzyme or protein of the invention can also include a nucleicacid molecule that hybridizes to a nucleic acid disclosed herein by SEQID NO, GenBank and/or GI number or a nucleic acid molecule thathybridizes to a nucleic acid molecule that encodes an amino acidsequence disclosed herein by SEQ ID NO, GenBank and/or GI number.Hybridization conditions can include highly stringent, moderatelystringent, or low stringency hybridization conditions that are wellknown to one of skill in the art such as those described herein.Similarly, a nucleic acid molecule that can be used in the invention canbe described as having a certain percent sequence identity to a nucleicacid disclosed herein by SEQ ID NO, GenBank and/or GI number or anucleic acid molecule that hybridizes to a nucleic acid molecule thatencodes an amino acid sequence disclosed herein by SEQ ID NO, GenBankand/or GI number. For example, the nucleic acid molecule can have atleast 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity to a nucleic acid described herein.

Stringent hybridization refers to conditions under which hybridizedpolynucleotides are stable. As known to those of skill in the art, thestability of hybridized polynucleotides is reflected in the meltingtemperature (T_(m)) of the hybrids. In general, the stability ofhybridized polynucleotides is a function of the salt concentration, forexample, the sodium ion concentration and temperature. A hybridizationreaction can be performed under conditions of lower stringency, followedby washes of varying, but higher, stringency. Reference to hybridizationstringency relates to such washing conditions. Highly stringenthybridization includes conditions that permit hybridization of onlythose nucleic acid sequences that form stable hybridized polynucleotidesin 0.018M NaCl at 65° C., for example, if a hybrid is not stable in0.018M NaCl at 65° C., it will not be stable under high stringencyconditions, as contemplated herein. High stringency conditions can beprovided, for example, by hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Hybridization conditions other than highlystringent hybridization conditions can also be used to describe thenucleic acid sequences disclosed herein. For example, the phrasemoderately stringent hybridization refers to conditions equivalent tohybridization in 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDSat 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C. Thephrase low stringency hybridization refers to conditions equivalent tohybridization in 10% formamide, 5×Denhart's solution, 6×SSPE, 0.2% SDSat 22° C., followed by washing in 1×SSPE, 0.2% SDS, at 37° C. Denhart'ssolution contains 1% Ficoll, 1% polyvinylpyrrolidone, and 1% bovineserum albumin (BSA). 20×SSPE (sodium chloride, sodium phosphate,ethylene diamide tetraacetic acid (EDTA)) contains 3M sodium chloride,0.2M sodium phosphate, and 0.025 M (EDTA). Other suitable low, moderateand high stringency hybridization buffers and conditions are well knownto those of skill in the art and are described, for example, in Sambrooket al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold SpringHarbor Laboratory, New York (2001); and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1999).

A nucleic acid molecule encoding an acetyl-CoA or a bioderived compoundpathway enzyme or protein of the invention can have at least a certainsequence identity to a nucleotide sequence disclosed herein. According,in some aspects of the invention, a nucleic acid molecule encoding anacetyl-CoA or a bioderived compound pathway enzyme or protein has anucleotide sequence of at least 65% identity, at least 70% identity, atleast 75% identity, at least 80% identity, at least 85% identity, atleast 90% identity, at least 91% identity, at least 92% identity, atleast 93% identity, at least 94% identity, at least 95% identity, atleast 96% identity, at least 97% identity, at least 98% identity, or atleast 99% identity to a nucleic acid disclosed herein by SEQ ID NO,GenBank and/or GI number or a nucleic acid molecule that hybridizes to anucleic acid molecule that encodes an amino acid sequence disclosedherein by SEQ ID NO, GenBank and/or GI number.

Sequence identity (also known as homology or similarity) refers tosequence similarity between two nucleic acid molecules or between twopolypeptides. Identity can be determined by comparing a position in eachsequence, which may be aligned for purposes of comparison. When aposition in the compared sequence is occupied by the same base or aminoacid, then the molecules are identical at that position. A degree ofidentity between sequences is a function of the number of matching orhomologous positions shared by the sequences. The alignment of twosequences to determine their percent sequence identity can be done usingsoftware programs known in the art, such as, for example, thosedescribed in Ausubel et al., Current Protocols in Molecular Biology,John Wiley and Sons, Baltimore, Md. (1999). Preferably, defaultparameters are used for the alignment. One alignment program well knownin the art that can be used is BLAST set to default parameters. Inparticular, programs are BLASTN and BLASTP, using the following defaultparameters: Genetic code=standard; filter=none; strand=both; cutoff=60;expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGHSCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR Details of these programs can befound at the National Center for Biotechnology Information.

Methods for constructing and testing the expression levels of anon-naturally occurring acetyl-CoA or a bioderived compound-producinghost can be performed, for example, by recombinant and detection methodswell known in the art. Such methods can be found described in, forexample, Sambrook et al., Molecular Cloning: A Laboratory Manual, ThirdEd., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al.,Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore,Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for production ofacetyl-CoA or a bioderived compound can be introduced stably ortransiently into a host cell using techniques well known in the artincluding, but not limited to, conjugation, electroporation, chemicaltransformation, transduction, transfection, and ultrasoundtransformation. For exogenous expression in E. coli or other prokaryoticcells, some nucleic acid sequences in the genes or cDNAs of eukaryoticnucleic acids can encode targeting signals such as an N-terminalmitochondrial or other targeting signal, which can be removed beforetransformation into prokaryotic host cells, if desired. For example,removal of a mitochondrial leader sequence led to increased expressionin E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)).For exogenous expression in yeast or other eukaryotic cells, genes canbe expressed in the cytosol without the addition of leader sequence, orcan be targeted to mitochondrion or other organelles, or targeted forsecretion, by the addition of a suitable targeting sequence such as amitochondrial targeting or secretion signal suitable for the host cells.Thus, it is understood that appropriate modifications to a nucleic acidsequence to remove or include a targeting sequence can be incorporatedinto an exogenous nucleic acid sequence to impart desirable properties.Furthermore, genes can be subjected to codon optimization withtechniques well known in the art to achieve optimized expression of theproteins.

An expression vector or vectors can be constructed to include one ormore acetyl-CoA or a bioderived compound biosynthetic pathway encodingnucleic acids as exemplified herein operably linked to expressioncontrol sequences functional in the host organism. Expression vectorsapplicable for use in the microbial host organisms of the inventioninclude, for example, plasmids, phage vectors, viral vectors, episomesand artificial chromosomes, including vectors and selection sequences ormarkers operable for stable integration into a host chromosome.Additionally, the expression vectors can include one or more selectablemarker genes and appropriate expression control sequences. Selectablemarker genes also can be included that, for example, provide resistanceto antibiotics or toxins, complement auxotrophic deficiencies, or supplycritical nutrients not in the culture media. Expression controlsequences can include constitutive and inducible promoters,transcription enhancers, transcription terminators, and the like whichare well known in the art. When two or more exogenous encoding nucleicacids are to be co-expressed, both nucleic acids can be inserted, forexample, into a single expression vector or in separate expressionvectors. For single vector expression, the encoding nucleic acids can beoperationally linked to one common expression control sequence or linkedto different expression control sequences, such as one induciblepromoter and one constitutive promoter. The transformation of exogenousnucleic acid sequences involved in a metabolic or synthetic pathway canbe confirmed using methods well known in the art. Such methods include,for example, nucleic acid analysis such as Northern blots or polymerasechain reaction (PCR) amplification of mRNA, or immunoblotting forexpression of gene products, or other suitable analytical methods totest the expression of an introduced nucleic acid sequence or itscorresponding gene product. It is understood by those skilled in the artthat the exogenous nucleic acid is expressed in a sufficient amount toproduce the desired product, and it is further understood thatexpression levels can be optimized to obtain sufficient expression usingmethods well known in the art and as disclosed herein.

In some embodiments, the invention also provides a method for producinga bioderived compound described herein. Such a method can compriseculturing the non-naturally occurring microbial organism as describedherein under conditions and for a sufficient period of time to producethe bioderived compound. In another embodiment, method further includesseparating the bioderived compound from other components in the culture.In this aspect, separating can include extraction, continuousliquid-liquid extraction, pervaporation, membrane filtration, membraneseparation, reverse osmosis, electrodialysis, distillation,crystallization, centrifugation, extractive filtration, ion exchangechromatography, absorption chromatography, or ultrafiltration.

In some embodiments, depending on the bioderived compound, the method ofthe invention may further include chemically converting a bioderivedcompound to the directed final compound. For example, in someembodiments, wherein the bioderived compound is butadiene, the method ofthe invention can further include chemically dehydrating 1,3-butanediol,crotyl alcohol, or 3-buten-2-ol to produce the butadiene.

Suitable purification and/or assays to test for the production ofacetyl-CoA or a bioderived compound can be performed using well knownmethods. Suitable replicates such as triplicate cultures can be grownfor each engineered strain to be tested. For example, product andbyproduct formation in the engineered production host can be monitored.The final product and intermediates, and other organic compounds, can beanalyzed by methods such as HPLC (High Performance LiquidChromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS(Liquid Chromatography-Mass Spectroscopy) or other suitable analyticalmethods using routine procedures well known in the art. The release ofproduct in the fermentation broth can also be tested with the culturesupernatant. Byproducts and residual glucose can be quantified by HPLCusing, for example, a refractive index detector for glucose andalcohols, and a UV detector for organic acids (Lin et al., Biotechnol.Bioeng. 90:775-779 (2005)), or other suitable assay and detectionmethods well known in the art. The individual enzyme or proteinactivities from the exogenous DNA sequences can also be assayed usingmethods well known in the art.

The bioderived compound can be separated from other components in theculture using a variety of methods well known in the art. Suchseparation methods include, for example, extraction procedures as wellas methods that include continuous liquid-liquid extraction,pervaporation, membrane filtration, membrane separation, reverseosmosis, electrodialysis, distillation, crystallization, centrifugation,extractive filtration, ion exchange chromatography, size exclusionchromatography, adsorption chromatography, and ultrafiltration. All ofthe above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the bioderived compound producers can becultured for the biosynthetic production of a bioderived compounddisclosed herein. Accordingly, in some embodiments, the inventionprovides culture medium having the bioderived compound or bioderivedcompound pathway intermediate described herein. In some aspects, theculture medium can also be separated from the non-naturally occurringmicrobial organisms of the invention that produced the bioderivedcompound or bioderived compound pathway intermediate. Methods forseparating a microbial organism from culture medium are well known inthe art. Exemplary methods include filtration, flocculation,precipitation, centrifugation, sedimentation, and the like.

For the production of acetyl-CoA or a bioderived compound, therecombinant strains are cultured in a medium with carbon source andother essential nutrients. It is sometimes desirable and can be highlydesirable to maintain anaerobic conditions in the fermenter to reducethe cost of the overall process. Such conditions can be obtained, forexample, by first sparging the medium with nitrogen and then sealing theflasks with a septum and crimp-cap. For strains where growth is notobserved anaerobically, microaerobic or substantially anaerobicconditions can be applied by perforating the septum with a small holefor limited aeration. Exemplary anaerobic conditions have been describedpreviously and are well-known in the art. Exemplary aerobic andanaerobic conditions are described, for example, in United Statepublication 2009/0047719, filed Aug. 10, 2007. Fermentations can beperformed in a batch, fed-batch or continuous manner, as disclosedherein. Fermentations can also be conducted in two phases, if desired.The first phase can be aerobic to allow for high growth and thereforehigh productivity, followed by an anaerobic phase of high acetyl-CoA ora bioderived compound yields.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

The growth medium, can include, for example, any carbohydrate sourcewhich can supply a source of carbon to the non-naturally occurringmicrobial organism of the invention. Such sources include, for example,sugars such as glucose, xylose, arabinose, galactose, mannose, fructose,sucrose and starch; or glycerol, alone as the sole source of carbon orin combination with other carbon sources described herein or known inthe art. In one embodiment, the carbon source is a sugar. In oneembodiment, the carbon source is a sugar-containing biomass. In someembodiments, the sugar is glucose. In one embodiment, the sugar isxylose. In another embodiment, the sugar is arabinose. In oneembodiment, the sugar is galactose. In another embodiment, the sugar isfructose. In other embodiments, the sugar is sucrose. In one embodiment,the sugar is starch. In certain embodiments, the carbon source isglycerol. In some embodiments, the carbon source is crude glycerol. Inone embodiment, the carbon source is crude glycerol without treatment.In other embodiments, the carbon source is glycerol and glucose. Inanother embodiment, the carbon source is methanol and glycerol. In oneembodiment, the carbon source is carbon dioxide. In one embodiment, thecarbon source is formate. In one embodiment, the carbon source ismethane. In one embodiment, the carbon source is methanol. In certainembodiments, methanol is used alone as the sole source of carbon or incombination with other carbon sources described herein or known in theart. In a specific embodiment, the methanol is the only (sole) carbonsource. In one embodiment, the carbon source is chemoelectro-generatedcarbon (see, e.g., Liao et al. (2012) Science 335:1596). In oneembodiment, the chemoelectro-generated carbon is methanol. In oneembodiment, the chemoelectro-generated carbon is formate. In oneembodiment, the chemoelectro-generated carbon is formate and methanol.In one embodiment, the carbon source is a carbohydrate and methanol. Inone embodiment, the carbon source is a sugar and methanol. In anotherembodiment, the carbon source is a sugar and glycerol. In otherembodiments, the carbon source is a sugar and crude glycerol. In yetother embodiments, the carbon source is a sugar and crude glycerolwithout treatment. In one embodiment, the carbon source is asugar-containing biomass and methanol. In another embodiment, the carbonsource is a sugar-containing biomass and glycerol. In other embodiments,the carbon source is a sugar-containing biomass and crude glycerol. Inyet other embodiments, the carbon source is a sugar-containing biomassand crude glycerol without treatment. In some embodiments, the carbonsource is a sugar-containing biomass, methanol and a carbohydrate. Othersources of carbohydrate include, for example, renewable feedstocks andbiomass. Exemplary types of biomasses that can be used as feedstocks inthe methods provided herein include cellulosic biomass, hemicellulosicbiomass and lignin feedstocks or portions of feedstocks. Such biomassfeedstocks contain, for example, carbohydrate substrates useful ascarbon sources such as glucose, xylose, arabinose, galactose, mannose,fructose and starch. Given the teachings and guidance provided herein,those skilled in the art will understand that renewable feedstocks andbiomass other than those exemplified above also can be used forculturing the microbial organisms provided herein for the production ofsuccinate and other pathway intermediates.

In one embodiment, the carbon source is glycerol. In certainembodiments, the glycerol carbon source is crude glycerol or crudeglycerol without further treatment. In a further embodiment, the carbonsource comprises glycerol or crude glycerol, and also sugar or asugar-containing biomass, such as glucose. In a specific embodiment, theconcentration of glycerol in the fermentation broth is maintained byfeeding crude glycerol, or a mixture of crude glycerol and sugar (e.g.,glucose). In certain embodiments, sugar is provided for sufficientstrain growth. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of glycerol to sugar of from200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of glycerol to sugar of from100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of glycerol to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of glycerol to sugar of from 50:1 to 5:1.In certain embodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 100:1. In one embodiment,the sugar (e.g, glucose) is provided at a molar concentration ratio ofglycerol to sugar of 90:1. In one embodiment, the sugar (e.g, glucose)is provided at a molar concentration ratio of glycerol to sugar of 80:1.In one embodiment, the sugar (e.g, glucose) is provided at a molarconcentration ratio of glycerol to sugar of 70:1. In one embodiment, thesugar (e.g, glucose) is provided at a molar concentration ratio ofglycerol to sugar of 60:1. In one embodiment, the sugar (e.g, glucose)is provided at a molar concentration ratio of glycerol to sugar of 50:1.In one embodiment, the sugar (e.g, glucose) is provided at a molarconcentration ratio of glycerol to sugar of 40:1. In one embodiment, thesugar (e.g, glucose) is provided at a molar concentration ratio ofglycerol to sugar of 30:1. In one embodiment, the sugar (e.g, glucose)is provided at a molar concentration ratio of glycerol to sugar of 20:1.In one embodiment, the sugar (e.g, glucose) is provided at a molarconcentration ratio of glycerol to sugar of 10:1. In one embodiment, thesugar (e.g, glucose) is provided at a molar concentration ratio ofglycerol to sugar of 5:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 2:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:1. In certain embodiments,the sugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 1:100. In one embodiment, the sugar (e.g, glucose)is provided at a molar concentration ratio of glycerol to sugar of 1:90.In one embodiment, the sugar (e.g, glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:80. In one embodiment, thesugar (e.g, glucose) is provided at a molar concentration ratio ofglycerol to sugar of 1:70. In one embodiment, the sugar (e.g, glucose)is provided at a molar concentration ratio of glycerol to sugar of 1:60.In one embodiment, the sugar (e.g, glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:50. In one embodiment, thesugar (e.g, glucose) is provided at a molar concentration ratio ofglycerol to sugar of 1:40. In one embodiment, the sugar (e.g, glucose)is provided at a molar concentration ratio of glycerol to sugar of 1:30.In one embodiment, the sugar (e.g, glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:20. In one embodiment, thesugar (e.g, glucose) is provided at a molar concentration ratio ofglycerol to sugar of 1:10. In one embodiment, the sugar (e.g, glucose)is provided at a molar concentration ratio of glycerol to sugar of 1:5.In one embodiment, the sugar (e.g, glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:2. In certain embodimentsof the ratios provided above, the sugar is a sugar-containing biomass.In certain other embodiments of the ratios provided above, the glycerolis a crude glycerol or a crude glycerol without further treatment. Inother embodiments of the ratios provided above, the sugar is asugar-containing biomass, and the glycerol is a crude glycerol or acrude glycerol without further treatment.

Crude glycerol can be a by-product produced in the production ofbiodiesel, and can be used for fermentation without any furthertreatment. Biodiesel production methods include (1) a chemical methodwherein the glycerol-group of vegetable oils or animal oils issubstituted by low-carbon alcohols such as methanol or ethanol toproduce a corresponding fatty acid methyl esters or fatty acid ethylesters by transesterification in the presence of acidic or basiccatalysts; (2) a biological method where biological enzymes or cells areused to catalyze transesterification reaction and the correspondingfatty acid methyl esters or fatty acid ethyl esters are produced; and(3) a supercritical method, wherein transesterification reaction iscarried out in a supercritical solvent system without any catalysts. Thechemical composition of crude glycerol can vary with the process used toproduce biodiesel, the transesterification efficiency, recoveryefficiency of the biodiesel, other impurities in the feedstock, andwhether methanol and catalysts were recovered. For example, the chemicalcompositions of eleven crude glycerol collected from seven Australianbiodiesel producers reported that glycerol content ranged between 38%and 96%, with some samples including more than 14% methanol and 29% ash.In certain embodiments, the crude glycerol comprises from 5% to 99%glycerol. In some embodiments, the crude glycerol comprises from 10% to90% glycerol. In some embodiments, the crude glycerol comprises from 10%to 80% glycerol. In some embodiments, the crude glycerol comprises from10% to 70% glycerol. In some embodiments, the crude glycerol comprisesfrom 10% to 60% glycerol. In some embodiments, the crude glycerolcomprises from 10% to 50% glycerol. In some embodiments, the crudeglycerol comprises from 10% to 40% glycerol. In some embodiments, thecrude glycerol comprises from 10% to 30% glycerol. In some embodiments,the crude glycerol comprises from 10% to 20% glycerol. In someembodiments, the crude glycerol comprises from 80% to 90% glycerol. Insome embodiments, the crude glycerol comprises from 70% to 90% glycerol.In some embodiments, the crude glycerol comprises from 60% to 90%glycerol. In some embodiments, the crude glycerol comprises from 50% to90% glycerol. In some embodiments, the crude glycerol comprises from 40%to 90% glycerol. In some embodiments, the crude glycerol comprises from30% to 90% glycerol. In some embodiments, the crude glycerol comprisesfrom 20% to 90% glycerol. In some embodiments, the crude glycerolcomprises from 20% to 40% glycerol. In some embodiments, the crudeglycerol comprises from 40% to 60% glycerol. In some embodiments, thecrude glycerol comprises from 60% to 80% glycerol. In some embodiments,the crude glycerol comprises from 50% to 70% glycerol. In oneembodiment, the glycerol comprises 5% glycerol. In one embodiment, theglycerol comprises 10% glycerol. In one embodiment, the glycerolcomprises 15% glycerol. In one embodiment, the glycerol comprises 20%glycerol. In one embodiment, the glycerol comprises 25% glycerol. In oneembodiment, the glycerol comprises 30% glycerol. In one embodiment, theglycerol comprises 35% glycerol. In one embodiment, the glycerolcomprises 40% glycerol. In one embodiment, the glycerol comprises 45%glycerol. In one embodiment, the glycerol comprises 50% glycerol. In oneembodiment, the glycerol comprises 55% glycerol. In one embodiment, theglycerol comprises 60% glycerol. In one embodiment, the glycerolcomprises 65% glycerol. In one embodiment, the glycerol comprises 70%glycerol. In one embodiment, the glycerol comprises 75% glycerol. In oneembodiment, the glycerol comprises 80% glycerol. In one embodiment, theglycerol comprises 85% glycerol. In one embodiment, the glycerolcomprises 90% glycerol. In one embodiment, the glycerol comprises 95%glycerol. In one embodiment, the glycerol comprises 99% glycerol.

In one embodiment, the carbon source is methanol or formate. In certainembodiments, methanol is used as a carbon source in a formaldehydefixation pathway provided herein. In one embodiment, the carbon sourceis methanol or formate. In other embodiments, formate is used as acarbon source in a formaldehyde fixation pathway provided herein. Inspecific embodiments, methanol is used as a carbon source in a methanoloxidation pathway provided herein, either alone or in combination withthe fatty alcohol, fatty aldehyde, fatty acid or isopropanol pathwaysprovided herein. In one embodiment, the carbon source is methanol. Inanother embodiment, the carbon source is formate.

In one embodiment, the carbon source comprises methanol, and sugar (e.g,glucose) or a sugar-containing biomass. In another embodiment, thecarbon source comprises formate, and sugar (e.g., glucose) or asugar-containing biomass. In one embodiment, the carbon source comprisesmethanol, formate, and sugar (e.g, glucose) or a sugar-containingbiomass. In specific embodiments, the methanol or formate, or both, inthe fermentation feed is provided as a mixture with sugar (e.g, glucose)or sugar-comprising biomass. In certain embodiments, sugar is providedfor sufficient strain growth.

In certain embodiments, the carbon source comprises methanol and a sugar(e.g, glucose). In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol to sugar of from200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol to sugar of from100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of methanol to sugar of from 50:1 to 5:1.In certain embodiments, the sugar (e.g, glucose) is provided at a molarconcentration ratio of methanol to sugar of 100:1. In one embodiment,the sugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 90:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 80:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 70:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 60:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 50:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 40:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 30:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 20:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 10:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 5:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 2:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:1. In certain embodiments,the sugar (e.g, glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:100. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:90.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:80. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:70. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:60.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:50. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:40. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:30.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:20. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:10. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:5.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:2. In certain embodimentsof the ratios provided above, the sugar is a sugar-containing biomass.

In certain embodiments, the carbon source comprises formate and a sugar(e.g., glucose). In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of from200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of from100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of formate to sugar of from 50:1 to 5:1.In certain embodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 100:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 90:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 80:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 70:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 60:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 50:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 40:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 30:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 20:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 10:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 5:1. In one embodiment, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of 2:1. Inone embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:1. In certain embodiments,the sugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:100. In one embodiment, the sugar (e.g, glucose)is provided at a molar concentration ratio of formate to sugar of 1:90.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:80. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:70. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:60.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:50. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:40. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:30.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:20. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:10. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:5.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:2. In certain embodimentsof the ratios provided above, the sugar is a sugar-containing biomass.

In certain embodiments, the carbon source comprises a mixture ofmethanol and formate, and a sugar (e.g., glucose). In certainembodiments, sugar is provided for sufficient strain growth. In someembodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of from 200:1 to1:200. In some embodiments, the sugar (e.g., glucose) is provided at amolar concentration ratio of methanol and formate to sugar of from 100:1to 1:100. In some embodiments, the sugar (e.g., glucose) is provided ata molar concentration ratio of methanol and formate to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of methanol and formate to sugar of from50:1 to 5:1. In certain embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol and formate to sugarof 100:1. In one embodiment, the sugar (e.g., glucose) is provided at amolar concentration ratio of methanol and formate to sugar of 90:1. Inone embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 80:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 70:1. In oneembodiment, the sugar (e.g, glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 60:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 50:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 40:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 30:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 20:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 10:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 5:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 2:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:1. In certainembodiments, the sugar (e.g, glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:100. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:90. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:80. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:70. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:60. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:50. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:40. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:30. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:20. In oneembodiment, the sugar (e.g, glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:10. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:5. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:2. In certainembodiments of the ratios provided above, the sugar is asugar-containing biomass.

In addition to renewable feedstocks such as those exemplified above, theacetyl-CoA or the bioderived compound producing microbial organisms ofthe invention also can be modified for growth on syngas as its source ofcarbon. In this specific embodiment, one or more proteins or enzymes areexpressed in the acetyl-CoA or the bioderived compound producingorganisms to provide a metabolic pathway for utilization of syngas orother gaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the majorproduct of gasification of coal and of carbonaceous materials such asbiomass materials, including agricultural crops and residues. Syngas isa mixture primarily of H2 and CO and can be obtained from thegasification of any organic feedstock, including but not limited tocoal, coal oil, natural gas, biomass, and waste organic matter.Gasification is generally carried out under a high fuel to oxygen ratio.Although largely H2 and CO, syngas can also include CO₂ and other gasesin smaller quantities. Thus, synthesis gas provides a cost effectivesource of gaseous carbon such as CO and, additionally, CO₂.

Accordingly, given the teachings and guidance provided herein, thoseskilled in the art will understand that a non-naturally occurringmicrobial organism can be produced that secretes the biosynthesizedcompounds of the invention when grown on a carbon source such as acarbohydrate. Such compounds include, for example, acetyl-CoA or abioderived compound and any of the intermediate metabolites in theacetyl-CoA or the bioderived compound pathway. All that is required isto engineer in one or more of the required enzyme or protein activitiesto achieve biosynthesis of the desired compound or intermediateincluding, for example, inclusion of some or all of the acetyl-CoA orthe bioderived compound biosynthetic pathways. Accordingly, theinvention provides a non-naturally occurring microbial organism thatproduces and/or secretes acetyl-CoA or a bioderived compound when grownon a carbohydrate or other carbon source and produces and/or secretesany of the intermediate metabolites shown in the acetyl-CoA or thebioderived compound pathway when grown on a carbohydrate or other carbonsource. The acetyl-CoA or the bioderived compound producing microbialorganisms of the invention can initiate synthesis from an intermediate,for example, F6P, E4P, formate, formyl-CoA, G3P, PYR, DHA, H6P, 3HBCOA,3HB, 3-hydroxybutyryl phosphate, 4-hydroxybutyrate,4-hydroxybutyryl-CoA, adipyl-CoA, adipate semialdehyde,3-hydroxyisobutyrate, or 2-hydroxyisobutyryl-CoA.

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding an acetyl-CoA ora bioderived compound pathway enzyme or protein in sufficient amounts toproduce acetyl-CoA or a bioderived compound. It is understood that themicrobial organisms of the invention are cultured under conditionssufficient to produce acetyl-CoA or a bioderived compound. Following theteachings and guidance provided herein, the non-naturally occurringmicrobial organisms of the invention can achieve biosynthesis of abioderived compound resulting in intracellular concentrations betweenabout 0.1-200 mM or more. Generally, the intracellular concentration ofa bioderived compound is between about 3-150 mM, particularly betweenabout 5-125 mM and more particularly between about 8-100 mM, includingabout 10 mM, 20 mM, 50 mM, 80 mM, or more. Intracellular concentrationsbetween and above each of these exemplary ranges also can be achievedfrom the non-naturally occurring microbial organisms of the invention.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. publication2009/0047719, filed Aug. 10, 2007. Any of these conditions can beemployed with the non-naturally occurring microbial organisms as well asother anaerobic conditions well known in the art. Under such anaerobicor substantially anaerobic conditions, the acetyl-CoA or the bioderivedcompound producers can synthesize a bioderived compound at intracellularconcentrations of 5-10 mM or more as well as all other concentrationsexemplified herein. It is understood that, even though the abovedescription refers to intracellular concentrations, acetyl-CoA or abioderived compound producing microbial organisms can produce abioderived compound intracellularly and/or secrete the product into theculture medium.

Exemplary fermentation processes include, but are not limited to,fed-batch fermentation and batch separation; fed-batch fermentation andcontinuous separation; and continuous fermentation and continuousseparation. In an exemplary batch fermentation protocol, the productionorganism is grown in a suitably sized bioreactor sparged with anappropriate gas. Under anaerobic conditions, the culture is sparged withan inert gas or combination of gases, for example, nitrogen, N2/CO₂mixture, argon, helium, and the like. As the cells grow and utilize thecarbon source, additional carbon source(s) and/or other nutrients arefed into the bioreactor at a rate approximately balancing consumption ofthe carbon source and/or nutrients. The temperature of the bioreactor ismaintained at a desired temperature, generally in the range of 22-37degrees C., but the temperature can be maintained at a higher or lowertemperature depending on the growth characteristics of the productionorganism and/or desired conditions for the fermentation process. Growthcontinues for a desired period of time to achieve desiredcharacteristics of the culture in the fermenter, for example, celldensity, product concentration, and the like. In a batch fermentationprocess, the time period for the fermentation is generally in the rangeof several hours to several days, for example, 8 to 24 hours, or 1, 2,3, 4 or 5 days, or up to a week, depending on the desired cultureconditions. The pH can be controlled or not, as desired, in which case aculture in which pH is not controlled will typically decrease to pH 3-6by the end of the run. Upon completion of the cultivation period, thefermenter contents can be passed through a cell separation unit, forexample, a centrifuge, filtration unit, and the like, to remove cellsand cell debris. In the case where the desired product is expressedintracellularly, the cells can be lysed or disrupted enzymatically orchemically prior to or after separation of cells from the fermentationbroth, as desired, in order to release additional product. Thefermentation broth can be transferred to a product separations unit.Isolation of product occurs by standard separations procedures employedin the art to separate a desired product from dilute aqueous solutions.Such methods include, but are not limited to, liquid-liquid extractionusing a water immiscible organic solvent (e.g, toluene or other suitablesolvents, including but not limited to diethyl ether, ethyl acetate,tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane,hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE),dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and thelike) to provide an organic solution of the product, if appropriate,standard distillation methods, and the like, depending on the chemicalcharacteristics of the product of the fermentation process.

In an exemplary fully continuous fermentation protocol, the productionorganism is generally first grown up in batch mode in order to achieve adesired cell density. When the carbon source and/or other nutrients areexhausted, feed medium of the same composition is supplied continuouslyat a desired rate, and fermentation liquid is withdrawn at the samerate. Under such conditions, the product concentration in the bioreactorgenerally remains constant, as well as the cell density. The temperatureof the fermenter is maintained at a desired temperature, as discussedabove. During the continuous fermentation phase, it is generallydesirable to maintain a suitable pH range for optimized production. ThepH can be monitored and maintained using routine methods, including theaddition of suitable acids or bases to maintain a desired pH range. Thebioreactor is operated continuously for extended periods of time,generally at least one week to several weeks and up to one month, orlonger, as appropriate and desired. The fermentation liquid and/orculture is monitored periodically, including sampling up to every day,as desired, to assure consistency of product concentration and/or celldensity. In continuous mode, fermenter contents are constantly removedas new feed medium is supplied. The exit stream, containing cells,medium, and product, are generally subjected to a continuous productseparations procedure, with or without removing cells and cell debris,as desired. Continuous separations methods employed in the art can beused to separate the product from dilute aqueous solutions, includingbut not limited to continuous liquid-liquid extraction using a waterimmiscible organic solvent (e.g., toluene or other suitable solvents,including but not limited to diethyl ether, ethyl acetate,tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane,hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE),dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and thelike), standard continuous distillation methods, and the like, or othermethods well known in the art.

In addition to the culturing and fermentation conditions disclosedherein, growth condition for achieving biosynthesis of acetyl-CoA or abioderived compound can include the addition of an osmoprotectant to theculturing conditions. In certain embodiments, the non-naturallyoccurring microbial organisms of the invention can be sustained,cultured or fermented as described herein in the presence of anosmoprotectant. Briefly, an osmoprotectant refers to a compound thatacts as an osmolyte and helps a microbial organism as described hereinsurvive osmotic stress. Osmoprotectants include, but are not limited to,betaines, amino acids, and the sugar trehalose. Non-limiting examples ofsuch are glycine betaine, praline betaine, dimethylthetin,dimethylslfonioproprionate, 3-dimethylsulfonio-2-methylproprionate,pipecolic acid, dimethylsulfonioacetate, choline, L-camitine andectoine. In one aspect, the osmoprotectant is glycine betaine. It isunderstood to one of ordinary skill in the art that the amount and typeof osmoprotectant suitable for protecting a microbial organism describedherein from osmotic stress will depend on the microbial organism used.The amount of osmoprotectant in the culturing conditions can be, forexample, no more than about 0.1 mM, no more than about 0.5 mM, no morethan about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM,no more than about 2.5 mM, no more than about 3.0 mM, no more than about5.0 mM, no more than about 7.0 mM, no more than about 10 mM, no morethan about 50 mM, no more than about 100 mM or no more than about 500mM.

In some embodiments, the carbon feedstock and other cellular uptakesources such as phosphate, ammonia, sulfate, chloride and other halogenscan be chosen to alter the isotopic distribution of the atoms present inacetyl-CoA or a bioderived compound or any acetyl-CoA or a bioderivedcompound pathway intermediate. The various carbon feedstock and otheruptake sources enumerated above will be referred to herein,collectively, as “uptake sources.” Uptake sources can provide isotopicenrichment for any atom present in the acetyl-CoA, bioderived compoundor pathway intermediate, or for side products generated in reactionsdiverging away from an acetyl-CoA or a bioderived compound pathway.Isotopic enrichment can be achieved for any target atom including, forexample, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus,chloride or other halogens.

In some embodiments, the uptake sources can be selected to alter thecarbon-12, carbon-13, and carbon-14 ratios. In some embodiments, theuptake sources can be selected to alter the oxygen-16, oxygen-17, andoxygen-18 ratios. In some embodiments, the uptake sources can beselected to alter the hydrogen, deuterium, and tritium ratios. In someembodiments, the uptake sources can be selected to alter the nitrogen-14and nitrogen-15 ratios. In some embodiments, the uptake sources can beselected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35ratios. In some embodiments, the uptake sources can be selected to alterthe phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In someembodiments, the uptake sources can be selected to alter thechlorine-35, chlorine-36, and chlorine-37 ratios.

In some embodiments, the isotopic ratio of a target atom can be variedto a desired ratio by selecting one or more uptake sources. An uptakesource can be derived from a natural source, as found in nature, or froma man-made source, and one skilled in the art can select a naturalsource, a man-made source, or a combination thereof, to achieve adesired isotopic ratio of a target atom. An example of a man-made uptakesource includes, for example, an uptake source that is at leastpartially derived from a chemical synthetic reaction. Such isotopicallyenriched uptake sources can be purchased commercially or prepared in thelaboratory and/or optionally mixed with a natural source of the uptakesource to achieve a desired isotopic ratio. In some embodiments, atarget atom isotopic ratio of an uptake source can be achieved byselecting a desired origin of the uptake source as found in nature. Forexample, as discussed herein, a natural source can be a biobased derivedfrom or synthesized by a biological organism or a source such aspetroleum-based products or the atmosphere. In some such embodiments, asource of carbon, for example, can be selected from a fossilfuel-derived carbon source, which can be relatively depleted ofcarbon-14, or an environmental or atmospheric carbon source, such asCO₂, which can possess a larger amount of carbon-14 than itspetroleum-derived counterpart.

The unstable carbon isotope carbon-14 or radiocarbon makes up forroughly 1 in 10¹² carbon atoms in the earth's atmosphere and has ahalf-life of about 5700 years. The stock of carbon is replenished in theupper atmosphere by a nuclear reaction involving cosmic rays andordinary nitrogen (¹⁴N). Fossil fuels contain no carbon-14, as itdecayed long ago. Burning of fossil fuels lowers the atmosphericcarbon-14 fraction, the so-called “Suess effect”.

Methods of determining the isotopic ratios of atoms in a compound arewell known to those skilled in the art. Isotopic enrichment is readilyassessed by mass spectrometry using techniques known in the art such asaccelerated mass spectrometry (AMS), Stable Isotope Ratio MassSpectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation byNuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques canbe integrated with separation techniques such as liquid chromatography(LC), high performance liquid chromatography (HPLC) and/or gaschromatography, and the like.

In the case of carbon, ASTM D6866 was developed in the United States asa standardized analytical method for determining the biobased content ofsolid, liquid, and gaseous samples using radiocarbon dating by theAmerican Society for Testing and Materials (ASTM) International. Thestandard is based on the use of radiocarbon dating for the determinationof a product's biobased content. ASTM D6866 was first published in 2004,and the current active version of the standard is ASTM D6866-11(effective Apr. 1, 2011). Radiocarbon dating techniques are well knownto those skilled in the art, including those described herein.

The biobased content of a compound is estimated by the ratio ofcarbon-14 (¹⁴C) to carbon-12 (¹²C). Specifically, the Fraction Modem(Fm) is computed from the expression: Fm=(S−B)/(M−B), where B, S and Mrepresent the ¹⁴C/¹²C ratios of the blank, the sample and the modemreference, respectively. Fraction Modem is a measurement of thedeviation of the ¹⁴C/¹²C ratio of a sample from “Modem.” Modem isdefined as 95% of the radiocarbon concentration (in AD 1950) of NationalBureau of Standards (NBS) Oxalic Acid I (i.e., standard referencematerials (SRM) 4990b) normalized to δ¹³C_(VPDB)=−19 per mil (Olsson,The use of Oxalic acid as a Standard. in, Radiocarbon Variations andAbsolute Chronology, Nobel Symposium, 12th Proc., John Wiley & Sons, NewYork (1970)). Mass spectrometry results, for example, measured by ASM,are calculated using the internationally agreed upon definition of 0.95times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalizedto δ¹³C_(VPDB)=−19 per mil. This is equivalent to an absolute (AD1950)¹⁴C/¹²C ratio of 1.176±0.010×10⁻¹² (Karlen et al., Arkiv Geoftsik,4:465-471 (1968)). The standard calculations take into account thedifferential uptake of one isotope with respect to another, for example,the preferential uptake in biological systems of C¹² over C¹³ over C″,and these corrections are reflected as a Fm corrected for δ¹³.

An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of1955 sugar beet. Although there were 1000 lbs made, this oxalic acidstandard is no longer commercially available. The Oxalic Acid IIstandard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of1977 French beet molasses. In the early 1980's, a group of 12laboratories measured the ratios of the two standards. The ratio of theactivity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). Theisotopic ratio of HOx II is −17.8 per mil. ASTM D6866-11 suggests use ofthe available Oxalic Acid II standard SRM 4990 C (Hox2) for the modemstandard (see discussion of original vs. currently available oxalic acidstandards in Mann, Radiocarbon, 25(2):519-527 (1983)). A Fm=0%represents the entire lack of carbon-14 atoms in a material, thusindicating a fossil (for example, petroleum based) carbon source. AFm=100%, after correction for the post-1950 injection of carbon-14 intothe atmosphere from nuclear bomb testing, indicates an entirely modemcarbon source. As described herein, such a “modem” source includesbiobased sources.

As described in ASTM D6866, the percent modem carbon (pMC) can begreater than 100% because of the continuing but diminishing effects ofthe 1950s nuclear testing programs, which resulted in a considerableenrichment of carbon-14 in the atmosphere as described in ASTM D6866-11.Because all sample carbon-14 activities are referenced to a “pre-bomb”standard, and because nearly all new biobased products are produced in apost-bomb environment, all pMC values (after correction for isotopicfraction) must be multiplied by 0.95 (as of 2010) to better reflect thetrue biobased content of the sample. A biobased content that is greaterthan 103% suggests that either an analytical error has occurred, or thatthe source of biobased carbon is more than several years old.

ASTM D6866 quantifies the biobased content relative to the material'stotal organic content and does not consider the inorganic carbon andother non-carbon containing substances present. For example, a productthat is 50% starch-based material and 50% water would be considered tohave a Biobased Content=100% (50% organic content that is 100% biobased)based on ASTM D6866. In another example, a product that is 50%starch-based material, 25% petroleum-based, and 25% water would have aBiobased Content=66.7% (75% organic content but only 50% of the productis biobased). In another example, a product that is 50% organic carbonand is a petroleum-based product would be considered to have a BiobasedContent=0% (50% organic carbon but from fossil sources). Thus, based onthe well known methods and known standards for determining the biobasedcontent of a compound or material, one skilled in the art can readilydetermine the biobased content and/or prepared downstream products thatutilize of the invention having a desired biobased content.

Applications of carbon-14 dating techniques to quantify bio-basedcontent of materials are known in the art (Currie et al., NuclearInstruments and Methods in Physics Research B, 172:281-287 (2000)). Forexample, carbon-14 dating has been used to quantify bio-based content interephthalate-containing materials (Colonna et al., Green Chemistry,13:2543-2548 (2011)). Notably, polypropylene terephthalate (PPT)polymers derived from renewable 1,3-propanediol and petroleum-derivedterephthalic acid resulted in Fm values near 30% (i.e., since 3/11 ofthe polymeric carbon derives from renewable 1,3-propanediol and 8/11from the fossil end member terephthalic acid) (Currie et al., supra,2000). In contrast, polybutylene terephthalate polymer derived from bothrenewable 1,4-butanediol and renewable terephthalic acid resulted inbio-based content exceeding 90% (Colonna et al., supra, 2011).

Accordingly, in some embodiments, the present invention providesacetyl-CoA or a bioderived compound or an acetyl-CoA or a bioderivedcompound pathway intermediate that has a carbon-12, carbon-13, andcarbon-14 ratio that reflects an atmospheric carbon, also referred to asenvironmental carbon, uptake source. For example, in some aspects theacetyl-CoA or the bioderived compound or an acetyl-CoA or a bioderivedcompound pathway intermediate can have an Fm value of at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98% or as much as 100%. In some suchembodiments, the uptake source is CO₂. In some embodiments, the presentinvention provides acetyl-CoA or a bioderived compound or an acetyl-CoAor a bioderived compound pathway intermediate that has a carbon-12,carbon-13, and carbon-14 ratio that reflects petroleum-based carbonuptake source. In this aspect, the acetyl-CoA or the bioderived compoundor an acetyl-CoA or a bioderived compound pathway intermediate can havean Fm value of less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, less than 50%, less than 45%, less than 40%, less than 35%,less than 30%, less than 25%, less than 20%, less than 15%, less than10%, less than 5%, less than 2% or less than 1%. In some embodiments,the present invention provides acetyl-CoA or a bioderived compound or anacetyl-CoA or a bioderived compound pathway intermediate that has acarbon-12, carbon-13, and carbon-14 ratio that is obtained by acombination of an atmospheric carbon uptake source with apetroleum-based uptake source. Using such a combination of uptakesources is one way by which the carbon-12, carbon-13, and carbon-14ratio can be varied, and the respective ratios would reflect theproportions of the uptake sources.

Further, the present invention relates to the biologically producedacetyl-CoA, a bioderived compound or pathway intermediate as disclosedherein, and to the products derived therefrom, wherein the acetyl-CoA orthe bioderived compound or an acetyl-CoA or a bioderived compoundpathway intermediate has a carbon-12, carbon-13, and carbon-14 isotoperatio of about the same value as the CO₂ that occurs in the environment.For example, in some aspects the invention provides bioderivedacetyl-CoA or a bioderived compound or a bioderived acetyl-CoA or abioderived compound intermediate having a carbon-12 versus carbon-13versus carbon-14 isotope ratio of about the same value as the CO₂ thatoccurs in the environment, or any of the other ratios disclosed herein.It is understood, as disclosed herein, that a product can have acarbon-12 versus carbon-13 versus carbon-14 isotope ratio of about thesame value as the CO₂ that occurs in the environment, or any of theratios disclosed herein, wherein the product is generated from abioderived compound or a bioderived compound pathway intermediate asdisclosed herein, wherein the bioderived product is chemically modifiedto generate a final product. Methods of chemically modifying abioderived product of a bioderived compound, or an intermediate thereof,to generate a desired product are well known to those skilled in theart, as described herein. The invention further provides biobasedproducts having a carbon-12 versus carbon-13 versus carbon-14 isotoperatio of about the same value as the CO₂ that occurs in the environment,wherein the biobased products are generated directly from or incombination with bioderived compound or a bioderived compound pathwayintermediate as disclosed herein.

Fatty alcohol, fatty aldehyde or fatty acid is a chemical used incommercial and industrial applications. Non-limiting examples of suchapplications include production of biofuels, chemicals, polymers,surfactants, soaps, detergents, shampoos, lubricating oil additives,fragrances, flavor materials and acrylates. Accordingly, in someembodiments, the invention provides biobased biofuels, chemicals,polymers, surfactants, soaps, detergents, shampoos, lubricating oiladditives, fragrances, flavor materials and acrylates comprising one ormore bioderived fatty alcohol, fatty aldehyde or fatty acid orbioderived fatty alcohol, fatty aldehyde or fatty acid pathwayintermediate produced by a non-naturally occurring microorganism of theinvention or produced using a method disclosed herein.

In some embodiments, the invention provides a biofuel, chemical,polymer, surfactant, soap, detergent, shampoo, lubricating oil additive,fragrance, flavor material or acrylate comprising bioderived fattyalcohol, fatty aldehyde or fatty acid or bioderived fatty alcohol, fattyaldehyde or fatty acid pathway intermediate, wherein the bioderivedfatty alcohol, fatty aldehyde or fatty acid or bioderived fatty alcohol,fatty aldehyde or fatty acid pathway intermediate includes all or partof the fatty alcohol, fatty aldehyde or fatty acid or fatty alcohol,fatty aldehyde or fatty acid pathway intermediate used in the productionof a biofuel, chemical, polymer, surfactant, soap, detergent, shampoo,lubricating oil additive, fragrance, flavor material or acrylate. Forexample, the final biofuel, chemical, polymer, surfactant, soap,detergent, shampoo, lubricating oil additive, fragrance, flavor materialor acrylate can contain the bioderived fatty alcohol, fatty aldehyde orfatty acid, fatty alcohol, fatty aldehyde or fatty acid pathwayintermediate, or a portion thereof that is the result of themanufacturing of the biofuel, chemical, polymer, surfactant, soap,detergent, shampoo, lubricating oil additive, fragrance, flavor materialor acrylate. Such manufacturing can include chemically reacting thebioderived fatty alcohol, fatty aldehyde or fatty acid, or bioderivedfatty alcohol, fatty aldehyde or fatty acid pathway intermediate (e.g.chemical conversion, chemical functionalization, chemical coupling,oxidation, reduction, polymerization, copolymerization and the like)with itself or another compound in a reaction that produces the finalbiofuel, chemical, polymer, surfactant, soap, detergent, shampoo,lubricating oil additive, fragrance, flavor material or acrylate. Thus,in some aspects, the invention provides a biobased biofuel, chemical,polymer, surfactant, soap, detergent, shampoo, lubricating oil additive,fragrance, flavor material or acrylate comprising at least 2%, at least3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98% or100% bioderived fatty alcohol, fatty aldehyde or fatty acid orbioderived fatty alcohol, fatty aldehyde or fatty acid pathwayintermediate as disclosed herein. In some aspects, when the product is abiobased polymer that includes or is obtained from a bioderived fattyalcohol, fatty aldehyde or fatty acid, or or fatty alcohol, fattyaldehyde or fatty acid pathway intermediate described herein, thebiobased polymer can be molded using methods well known in the art.Accordingly, in some embodiments, provided herein is a molded productcomprising the biobased polymer described herein.

Butadiene is a chemical commonly used in many commercial and industrialapplications. Provided herein are a bioderived butadiene and biobasedproducts comprising one or more bioderived butadiene or bioderivedbutadiene intermediate produced by a non-naturally occurringmicroorganism of the invention or produced using a method disclosedherein. Also provided herein are uses for bioderived butadiene and thebiobased products. Non-limiting examples are described herein andinclude the following. Biobased products comprising all or a portion ofbioderived butadiene include polymers, including synthetic rubbers andABS resins, and chemicals, including hexamethylenediamine (HMDA),1,4-butanediol, tetrahydrofuran (THF), adiponitrile, lauryl lactam,caprolactam, chloroprene, sulfalone, n-octanol and octene-1. Thebiobased polymers, including co-polymers, and resins include those wherebutadiene is reacted with one or more other chemicals, such as otheralkenes, e.g. styrene, to manufacture numerous copolymers, includingacrylonitrile 1,3-butadiene styrene (ABS), styrene-1,3-butadiene rubber(styrene butadiene rubber; SBR), styrene-1,3-butadiene latex. Productscomprising biobased butadiene in the form of polymer synthetic rubber(SBR) include synthetic rubber articles, including tires, adhesives,seals, sealants, coatings, hose and shoe soles, and in the form ofsynthetic rubber polybutadiene (polybutadiene rubber, PBR or BR) whichis used in synthetic rubber articles including tires, seals, gaskets andadhesives and as an intermediate in production of thermoplastic resinincluding acrylonitrile-butadiene-styrene (ABS) and in production ofhigh impact modifier of polymers such as high impact polystyrene (HIPS).ABS is used in molded articles, including pipe, telephone, computercasings, mobile phones, radios, and appliances. Other biobased BDpolymers include a latex, including styrene-butadiene latex (SB), usedfor example in paper coatings, carpet backing, adhesives, and foammattresses; nitrile rubber, used in for example hoses, fuel lines,gasket seals, gloves and footwear; and styrene-butadiene blockcopolymers, used for example in asphalt modifiers (for road and roofingconstruction applications), adhesives, footwear and toys. Chemicalintermediates made from butadiene include adiponitrile, HMDA, lauryllactam, and caprolactam, used for example in production of nylon,including nylon-6,6 and other nylon-6,X, and chloroprene used forexample in production of polychloroprene (neoprene). Butanediol producedfrom butadiene is used for example in production of speciality polymerresins including thermoplastic including polybutylene terephthalate(PBT), used in molded articles including parts for automotive,electrical, water systems and small appliances. Butadiene is also aco-monomer for polyurethane and polyurethane-polyurea copolymers.Butadiene is a co-monomer for biodegradable polymers, including PBAT(poly(butylene adipate-co-terephthalate)) and PBS (poly(butylenesuccinate)). Tetrahydrofuran produced from butadiene finds use as asolvent and in production of elastic fibers. Conversion of butadiene toTHF, and subsequently to polytetramethylene ether glycol (PTMEG) (alsoreferred to as PTMO, polytetramethylene oxide and PTHF,poly(tetrahydrofuran)), provides an intermediate used to manufactureelastic fibers, e.g. spandex fiber, used in products such as LYCRA®fibers or elastane, for example when combined with polyurethane-polyureacopolymers. THF also finds use as an industrial solvent and inpharmaceutical production. PTMEG is also combined with in the productionof specialty thermoplastic elastomers (TPE), including thermoplasticelastomer polyester (TPE-E or TPEE) and copolyester ethers (COPE). COPESare high modulus elastomers with excellent mechanical properties andoil/environmental resistance, allowing them to operate at high and lowtemperature extremes. PTMEG and butadiene also make thermoplasticpolyurethanes (e.g. TPE-U or TPEU) processed on standard thermoplasticextrusion, calendaring, and molding equipment, and are characterized bytheir outstanding toughness and abrasion resistance. Other biobasedproducts of bioderived BD include styrene block copolymers used forexample in bitumen modification, footwear, packaging, and moldedextruded products; methylmethacrylate butadiene styrene and methacrylatebutadiene styrene (MBS) resins—clear resins—used as impact modifier fortransparent thermoplastics including polycarbonate (PC), polyvinylcarbonate (PVC) and poly)methyl methacrylate (PMMA); sulfalone used as asolvent or chemical; n-octanol and octene-1. Accordingly, in someembodiments, the invention provides a biobased product comprising one ormore bioderived butadiene or bioderived butadiene intermediate producedby a non-naturally occurring microorganism of the invention or producedusing a method disclosed herein.

Crotyl alcohol, also referred to as 2-buten-1-ol, is a valuable chemicalintermediate. Crotyl alcohol is a chemical commonly used in manycommercial and industrial applications. Non-limiting examples of suchapplications include production of crotyl halides, esters, and ethers,which in turn are chemical are chemical intermediates in the productionof monomers, fine chemicals, such as sorbic acid, trimethylhydroquinone,crotonic acid and 3-methoxybutanol, agricultural chemicals, andpharmaceuticals. Exemplary fine chemical products include sorbic acid,trimethylhydroquinone, crotonic acid and 3-methoxybutanol. Crotylalcohol is also a precursor to 1,3-butadiene. Crotyl alcohol iscurrently produced exclusively from petroleum feedstocks. For exampleJapanese Patent 47-013009 and U.S. Pat. Nos. 3,090,815, 3,090,816, and3,542,883 describe a method of producing crotyl alcohol by isomerizationof 1,2-epoxybutane. The ability to manufacture crotyl alcohol fromalternative and/or renewable feedstocks would represent a major advancein the quest for more sustainable chemical production processes.Accordingly, in some embodiments, the invention provides a biobasedmonomer, fine chemical, agricultural chemical, or pharmaceuticalcomprising one or more bioderived crotyl alcohol or bioderived crotylalcohol intermediate produced by a non-naturally occurring microorganismof the invention or produced using a method disclosed herein.

1,3-Butanediol is a chemical commonly used in many commercial andindustrial applications. Non-limiting examples of such applicationsinclude its use as an organic solvent for food flavoring agents or as ahypoglycaemic agent and its use in the production of polyurethane andpolyester resins. Moreover, optically active 1,3-butanediol is also usedin the synthesis of biologically active compounds and liquid crystals.Still further, 1,3-butanediol can be used in commercial production of1,3-butadiene, a compound used in the manufacture of synthetic rubbers(e.g., tires), latex, and resins. 1,3-butanediol can also be sued tosynthesize (R)-3-hydroxybutyryl-(R)-1,3-butanediol monoester or(R)-3-ketobutyryl-(R)-1,3-butanediol. Accordingly, in some embodiments,the invention provides a biobased organic solvent, hypoglycaemic agent,polyurethane, polyester resin, synthetic rubber, latex, or resincomprising one or more bioderived 1,3-butanediol or bioderived1,3-butanediol intermediate produced by a non-naturally occurringmicroorganism of the invention or produced using a method disclosedherein.

3-Buten-2-ol is a chemical commonly used in many commercial andindustrial applications. Non-limiting examples of such applicationsinclude it use as a solvent, e.g. as a viscosity adjustor, a monomer forpolymer production, or a precursor to a fine chemical such as inproduction of contrast agents for imaging (see US20110091374) orproduction of glycerol (see US20120302800A1). 3-Buten-2-ol can also beused as a precursor in the production of 1,3-butadiene. Accordingly, insome embodiments, the invention provides a biobased solvent, polymer (orplastic or resin made from that polymer), or fine chemical comprisingone or more bioderived 3-buten-2-ol or bioderived 3-buten-2-olintermediate produced by a non-naturally occurring microorganism of theinvention or produced using a method disclosed herein.

In some embodiments, the invention provides polymer, synthetic rubber,resin, or chemical comprising bioderived butadiene or bioderivedbutadiene pathway intermediate, wherein the bioderived butadiene orbioderived butadiene pathway intermediate includes all or part of thebutadiene or butadiene pathway intermediate used in the production ofpolymer, synthetic rubber, resin, or chemical, or other biobasedproducts described herein (for example hexamethylenediamine (HMDA),1,4-butanediol, tetrahydrofuran (THF), adiponitrile, lauryl lactam,caprolactam, chloroprene, sulfalone, n-octanol, octene-1, ABS, SBR, PBR,PTMEG, COPE). Thus, in some aspects, the invention provides a biobasedpolymer, synthetic rubber, resin, or chemical or other biobased productdescribed herein comprising at least 2%, at least 3%, at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 98% or 100% bioderivedbutadiene or bioderived butadiene pathway intermediate as disclosedherein. Additionally, in some aspects, the invention provides a biobasedpolymer, synthetic rubber, resin, or chemical or other biobased productdescribed herein (for example hexamethylenediamine (HMDA),1,4-butanediol, tetrahydrofuran (THF), adiponitrile, lauryl lactam,caprolactam, chloroprene, sulfalone, n-octanol, octene-1, ABS, SBR, PBR,PTMEG, COPE), wherein the butadiene or butadiene pathway intermediateused in its production is a combination of bioderived and petroleumderived butadiene or butadiene pathway intermediate. For example, abiobased polymer, synthetic rubber, resin, or chemical or other biobasedproduct described herein (for example hexamethylenediamine (HMDA),1,4-butanediol, tetrahydrofuran (THF), adiponitrile, lauryl lactam,caprolactam, chloroprene, sulfalone, n-octanol, octene-1, ABS, SBR, PBR,PTMEG, COPE) can be produced using 50% bioderived butadiene and 50%petroleum derived butadiene or other desired ratios such as 60%/40%,70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%,10%/90% of bioderived/petroleum derived precursors, so long as at leasta portion of the product comprises a bioderived product produced by themicrobial organisms disclosed herein. It is understood that methods forproducing polymer, synthetic rubber, resin, or chemical or otherbiobased product described herein (for example hexamethylenediamine(HMDA), 1,4-butanediol, tetrahydrofuran (THF), adiponitrile, lauryllactam, caprolactam, chloroprene, sulfalone, n-octanol, octene-1, ABS,SBR, PBR, PTMEG, COPE) using the bioderived butadiene or bioderivedbutadiene pathway intermediate of the invention are well known in theart.

In some embodiments, the invention provides organic solvent,hypoglycaemic agent, polyurethane, polyester resin, synthetic rubber,latex, or resin comprising bioderived 1,3-butanediol or bioderived1,3-butanediol pathway intermediate, wherein the bioderived1,3-butanediol or bioderived 1,3-butanediol pathway intermediateincludes all or part of the 1,3-butanediol or 1,3-butanediol pathwayintermediate used in the production of organic solvent, hypoglycaemicagent, polyurethane, polyester resin, synthetic rubber, latex, or resin.Thus, in some aspects, the invention provides a biobased organicsolvent, hypoglycaemic agent, polyurethane, polyester resin, syntheticrubber, latex, or resin comprising at least 2%, at least 3%, at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 98% or 100%bioderived 1,3-butanediol or bioderived 1,3-butanediol pathwayintermediate as disclosed herein. Additionally, in some aspects, theinvention provides a biobased organic solvent, hypoglycaemic agent,polyurethane, polyester resin, synthetic rubber, latex, or resin whereinthe 1,3-butanediol or 1,3-butanediol pathway intermediate used in itsproduction is a combination of bioderived and petroleum derived1,3-butanediol or 1,3-butanediol pathway intermediate. For example, abiobased organic solvent, hypoglycaemic agent, polyurethane, polyesterresin, synthetic rubber, latex, or resin can be produced using 50%bioderived 1,3-butanediol and 50% petroleum derived 1,3-butanediol orother desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%,100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleumderived precursors, so long as at least a portion of the productcomprises a bioderived product produced by the microbial organismsdisclosed herein. It is understood that methods for producing organicsolvent, hypoglycaemic agent, polyurethane, polyester resin, syntheticrubber, latex, or resin using the bioderived 1,3-butanediol orbioderived 1,3-butanediol pathway intermediate of the invention are wellknown in the art.

In some embodiments, the invention provides monomer, fine chemical,agricultural chemical, or pharmaceutical comprising bioderived crotylalcohol or bioderived crotyl alcohol pathway intermediate, wherein thebioderived crotyl alcohol or bioderived crotyl alcohol pathwayintermediate includes all or part of the crotyl alcohol or crotylalcohol pathway intermediate used in the production of monomer, finechemical, agricultural chemical, or pharmaceutical. Thus, in someaspects, the invention provides a biobased monomer, fine chemical,agricultural chemical, or pharmaceutical comprising at least 2%, atleast 3%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98% or 100% bioderived crotyl alcohol or bioderived crotyl alcoholpathway intermediate as disclosed herein. Additionally, in some aspects,the invention provides a biobased monomer, fine chemical, agriculturalchemical, or pharmaceutical wherein the crotyl alcohol or crotyl alcoholpathway intermediate used in its production is a combination ofbioderived and petroleum derived crotyl alcohol or crotyl alcoholpathway intermediate. For example, a biobased monomer, fine chemical,agricultural chemical, or pharmaceutical can be produced using 50%bioderived crotyl alcohol and 50% petroleum derived crotyl alcohol orother desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%,100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleumderived precursors, so long as at least a portion of the productcomprises a bioderived product produced by the microbial organismsdisclosed herein. It is understood that methods for producing monomer,fine chemical, agricultural chemical, or pharmaceutical using thebioderived crotyl alcohol or bioderived crotyl alcohol pathwayintermediate of the invention are well known in the art.

In some embodiments, the invention provides solvent (orsolvent-containing composition), polymer (or plastic or resin made fromthat polymer), or a fine chemical, comprising bioderived 3-buten-2-ol orbioderived 3-buten-2-ol pathway intermediate, wherein the bioderived3-buten-2-ol or bioderived 3-buten-2-ol pathway intermediate includesall or part of the 3-buten-2-ol or 3-buten-2-ol pathway intermediateused in the production of the solvent (or composition containing thesolvent), polymer (or plastic or resin made from that polymer) or finechemical. Thus, in some aspects, the invention provides a biobasedsolvent (or composition containing the solvent), polymer (or plastic orresin made from that polymer) or fine chemical comprising at least 2%,at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30% at least 35%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98% or 100% bioderived 3-buten-2-ol or bioderived 3-buten-2-olpathway intermediate as disclosed herein. Additionally, in some aspects,the invention provides the biobased solvent (or composition containingthe solvent), polymer (or plastic or resin made from that polymer) orfine chemical wherein the 3-buten-2-ol or 3-buten-2-ol pathwayintermediate used in its production is a combination of bioderived andpetroleum derived 3-buten-2-ol or 3-buten-2-ol pathway intermediate. Forexample, the biobased the solvent (or composition containing thesolvent), polymer (or plastic or resin made from that polymer) or finechemical can be produced using 50% bioderived 3-buten-2-ol and 50%petroleum derived 3-buten-2-ol or other desired ratios such as 60%/40%,70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%,10%/90% of bioderived/petroleum derived precursors, so long as at leasta portion of the product comprises a bioderived product produced by themicrobial organisms disclosed herein. It is understood that methods forproducing the solvent (or composition containing the solvent), polymer(or plastic or resin made from that polymer) or fine chemical using thebioderived 3-buten-2-ol or bioderived 3-buten-2-ol pathway intermediateof the invention are well known in the art.

1,4-Butanediol and/or 4-HB are chemicals used in commercial andindustrial applications. Non-limiting examples of such applicationsinclude production of plastics, elastic fibers, polyurethanes,polyesters, including polyhydroxyalkanoates such as P4HB or co-polymersthereof, PTMEG and polyurethane-polyurea copolymers, referred to asspandex, elastane or Lycra™, nylons, and the like. Moreover,1,4-butanediol and/or 4-HB are also used as a raw material in theproduction of a wide range of products including plastics, elasticfibers, polyurethanes, polyesters, including polyhydroxyalkanoates suchas P4HB or co-polymers thereof, PTMEG and polyurethane-polyureacopolymers, referred to as spandex, elastane or Lycra™, nylons, and thelike. Accordingly, in some embodiments, provided are biobased plastics,elastic fibers, polyurethanes, polyesters, includingpolyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG andpolyurethane-polyurea copolymers, referred to as spandex, elastane orLycra™, nylons, and the like, comprising one or more bioderived1,4-butanediol and/or 4-HB or bioderived 1,4-butanediol and/or 4-HBintermediate thereof produced by a non-naturally occurring microbialorganism provided herein or produced using a method disclosed herein.

In some embodiments, the invention provides plastics, elastic fibers,polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HBor co-polymers thereof, PTMEG and polyurethane-polyurea copolymers,referred to as spandex, elastane or Lycra™, nylons, and the like,comprising bioderived 1,4-butanediol and/or 4-HB or bioderived1,4-butanediol and/or 4-HB intermediate thereof, wherein the bioderived1,4-butanediol and/or 4-HB or bioderived 1,4-butanediol and/or 4-HBintermediate thereof includes all or part of the 1,4-butanediol and/or4-HB or 1,4-butanediol and/or 4-HB intermediate thereof used in theproduction of plastics, elastic fibers, polyurethanes, polyesters,including polyhydroxyalkanoates such as P4HB or co-polymers thereof,PTMEG and polyurethane-polyurea copolymers, referred to as spandex,elastane or Lycra™, nylons, and the like. Thus, in some aspects, theinvention provides a biobased plastics, elastic fibers, polyurethanes,polyesters, including polyhydroxyalkanoates such as P4HB or co-polymersthereof, PTMEG and polyurethane-polyurea copolymers, referred to asspandex, elastane or Lycra™, nylons, and the like, comprising at least2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80% at least 90%, at least 95%, atleast 98% or 100% bioderived 1,4-butanediol and/or 4-HB or bioderived1,4-butanediol and/or 4-HB intermediate thereof as disclosed herein.

In one embodiment, the product is a plastic. In one embodiment, theproduct is an elastic fiber. In one embodiment, the product is apolyurethane. In one embodiment, the product is a polyester. In oneembodiment, the product is a polyhydroxyalkanoate. In one embodiment,the product is a poly-4-HB. In one embodiment, the product is aco-polymer of poly-4-HB. In one embodiment, the product is apoly(tetramethylene ether) glycol. In one embodiment, the product is apolyurethane-polyurea copolymer. In one embodiment, the product is aspandex. In one embodiment, the product is an elastane. In oneembodiment, the product is a Lycra™. In one embodiment, the product is anylon.

Adipate, 6-aminocaproate, hexamethylenediamine and caprolactam, as wellas intermediates thereof, are chemicals used in commercial andindustrial applications. Non-limiting examples of such applicationsinclude production of polymers, plastics, epoxy resins, nylons (e.g.,nylon-6 or nylon 6-6), textiles, polyurethanes, plasticizers,unsaturated polyesters, fibers, polyester polyols, polyurethane,lubricant components, PVC, food additives, food ingredients, flavorants,gelling aids, food and oral medicinal coatings/products, and the like.Moreover, adipate, 6-aminocaproate, hexamethylenediamine and caprolactamare also used as a raw material in the production of a wide range ofproducts including polymers, plastics, epoxy resins, nylons (e.g.,nylon-6 or nylon 6-6), textiles, polyurethanes, plasticizers,unsaturated polyesters, fibers, polyester polyols, polyurethane,lubricant components, PVC, food additives, food ingredients, flavorants,gelling aids, food and oral medicinal coatings/products, and the like.Accordingly, in some embodiments, provided is biobased polymers,plastics, epoxy resins, nylons (e.g., nylon-6 or nylon 6-6), textiles,polyurethanes, plasticizers, unsaturated polyesters, fibers, polyesterpolyols, polyurethane, lubricant components, PVC, food additives, foodingredients, flavorants, gelling aids, food and oral medicinalcoatings/products, and the like, comprising one or more of bioderivedadipate, 6-aminocaproate, hexamethylenediamine or caprolactam, or abioderived intermediate thereof, produced by a non-naturally occurringmicrobial organism provided herein or produced using a method disclosedherein.

In one embodiment, the product is a polymer. In one embodiment, theproduct is a plastic. In one embodiment, the product is an epoxy resin.In one embodiment, the product is a nylons (e.g., nylon-6 or nylon 6-6).In one embodiment, the product is a textile. In one embodiment, theproduct is a polyurethane. In one embodiment, the product is aplasticizer. In one embodiment, the product is an unsaturated polyester.In one embodiment, the product is a fiber. In one embodiment, theproduct is a polyester polyol. In one embodiment, the product is apolyurethane. In one embodiment, the product is a lubricant component.In one embodiment, the product is a PVC. In one embodiment, the productis a food additive. In one embodiment, the product is a food ingredient.In one embodiment, the product is a flavorant. In one embodiment, theproduct is a gelling aid. In one embodiment, the product is a foodcoating. In one embodiment, the product is a food product. In oneembodiment, the product is an oral medicinal coatings. In oneembodiment, the product is an oral product

In some embodiments, provided is polymers, plastics, epoxy resins,nylons (e.g., nylon-6 or nylon 6-6), textiles, polyurethanes,plasticizers, unsaturated polyesters, fibers, polyester polyols,polyurethane, lubricant components, PVC, food additives, foodingredients, flavorants, gelling aids, food and oral medicinalcoatings/products, and the like, comprising bioderived adipate,6-aminocaproate, hexamethylenediamine or caprolactam, or a bioderivedintermediate thereof, wherein the bioderived adipate, 6-aminocaproate,hexamethylenediamine or caprolactam, or bioderived intermediate thereof,includes all or part of an adipate, 6-aminocaproate,hexamethylenediamine or caprolactam, or an intermediate thereof, used inthe production of polymers, plastics, epoxy resins, nylons (e.g.,nylon-6 or nylon 6-6), textiles, polyurethanes, plasticizers,unsaturated polyesters, fibers, polyester polyols, polyurethane,lubricant components, PVC, food additives, food ingredients, flavorants,gelling aids, food and oral medicinal coatings/products, and the like.Thus, in some aspects, provided is a biobased polymers, plastics, epoxyresins, nylons (e.g., nylon-6 or nylon 6-6), textiles, polyurethanes,plasticizers, unsaturated polyesters, fibers, polyester polyols,polyurethane, lubricant components, PVC, food additives, foodingredients, flavorants, gelling aids, food and oral medicinalcoatings/products, and the like, comprising at least 2%, at least 3%, atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98% or100% bioderived adipate, 6-aminocaproate, hexamethylenediamine orcaprolactam, or a bioderived adipate, 6-aminocaproate,hexamethylenediamine or caprolactam intermediate, as disclosed herein.

Methacylic acid, as well as intermediates thereof such as3-hydroxyisobutyrate, and 2-hydroxyisobutyric acid is a chemical used incommercial and industrial applications. Non-limiting examples of suchapplications include production of polymers, co-polymers, plastics,methacrylates (e.g., a methyl methacrylate or a butyl methacrylate),glacial methacylic acid, and the like. 2-Hydroxyisobutyric acid can bedehydrated to form methacrylic acid as described, for example, in U.S.Pat. No. 7,186,856. Moreover, 3-hydroxyisobutyrate and methacylic acidare also used as a raw material in the production of a wide range ofproducts including polymers, co-polymers, plastics, methacrylates (e.g.,a methyl methacrylate or a butyl methacrylate), glacial methacylic acid,and the like. Accordingly, in some embodiments, the invention providesbiobased polymers, co-polymers, plastics, methacrylates (e.g., a methylmethacrylate or a butyl methacrylate), glacial methacylic acid, and thelike, comprising one or more of bioderived methacylic acid,3-hydroxyisobutyrate or 2-hydroxyisobutyric acid, or a bioderivedintermediate thereof, produced by a non-naturally occurringmicroorganism of the invention or produced using a method disclosedherein.

In some embodiments, the invention provides polymers, co-polymers,plastics, methacrylates (e.g., a methyl methacrylate or a butylmethacrylate), glacial methacylic acid, and the like, comprisingbioderived methacylic acid, 3-hydroxyisobutyrate or 2-hydroxyisobutyricacid, or a bioderived intermediate thereof, wherein the bioderivedmethacylic acid, 3-hydroxyisobutyrate or 2-hydroxyisobutyric acid, orbioderived intermediate thereof, includes all or part of the amethacylic acid, 3-hydroxyisobutyrate or 2-hydroxyisobutyric acid, or anintermediate thereof, used in the production of polymers, co-polymers,plastics, methacrylates (e.g., a methyl methacrylate or a butylmethacrylate), glacial methacylic acid, and the like. Thus, in someaspects, the invention provides a biobased polymers, co-polymers,plastics, methacrylates (e.g., a methyl methacrylate or a butylmethacrylate), glacial methacylic acid, and the like, comprising atleast 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98% or 100% bioderived methacylic acid,3-hydroxyisobutyrate or 2-hydroxyisobutyric acid, or a bioderivedmethacylic acid, 3-hydroxyisobutyrate or 2-hydroxyisobutyric acidintermediate, as disclosed herein.

Additionally, in some embodiments, the invention provides a compositionhaving a bioderived compound or pathway intermediate disclosed hereinand a compound other than the bioderived compound or pathwayintermediate. For example, in some aspects, the invention provides abiobased product as described herein wherein the bioderived compound orbioderived compound pathway intermediate used in its production is acombination of bioderived and petroleum derived compound or compoundpathway intermediate. For example, a biobased product described hereincan be produced using 50% bioderived compound and 50% petroleum derivedcompound or other desired ratios such as 60%/40%, 70%/30%, 80%/20%,90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% ofbioderived/petroleum derived precursors, so long as at least a portionof the product comprises a bioderived product produced by the microbialorganisms disclosed herein. It is understood that methods for producinga biobased product as described herein using the bioderived compound orbioderived compound pathway intermediate of the invention are well knownin the art.

The invention further provides a composition comprising bioderivedcompound described herein and a compound other than the bioderivedbioderived. The compound other than the bioderived product can be acellular portion, for example, a trace amount of a cellular portion of,or can be fermentation broth or culture medium, or a purified orpartially purified fraction thereof produced in the presence of, anon-naturally occurring microbial organism of the invention. Thecomposition can comprise, for example, a reduced level of a byproductwhen produced by an organism having reduced byproduct formation, asdisclosed herein. The composition can comprise, for example, bioderivedcompound, or a cell lysate or culture supernatant of a microbialorganism of the invention.

In certain embodiments, provided herein is a composition comprising abioderived compound provided herein produced by culturing anon-naturally occurring microbial organism described herein. In someembodiments, the composition further comprises a compound other thansaid bioderived compound. In certain embodiments, the compound otherthan said bioderived compound is a trace amount of a cellular portion ofa non-naturally occurring microbial organism describe herein.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of acetyl-CoA or a bioderived compound includes anaerobicculture or fermentation conditions. In certain embodiments, thenon-naturally occurring microbial organisms of the invention can besustained, cultured or fermented under anaerobic or substantiallyanaerobic conditions. Briefly, an anaerobic condition refers to anenvironment devoid of oxygen. Substantially anaerobic conditionsinclude, for example, a culture, batch fermentation or continuousfermentation such that the dissolved oxygen concentration in the mediumremains between 0 and 10% of saturation. Substantially anaerobicconditions also includes growing or resting cells in liquid medium or onsolid agar inside a sealed chamber maintained with an atmosphere of lessthan 1% oxygen. The percent of oxygen can be maintained by, for example,sparging the culture with an N₂/CO₂ mixture or other suitable non-oxygengas or gases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of acetyl-CoA or a bioderived compound.Exemplary growth procedures include, for example, fed-batch fermentationand batch separation; fed-batch fermentation and continuous separation,or continuous fermentation and continuous separation. All of theseprocesses are well known in the art. Fermentation procedures areparticularly useful for the biosynthetic production of commercialquantities of acetyl-CoA or a bioderived compound. Generally, and aswith non-continuous culture procedures, the continuous and/ornear-continuous production of acetyl-CoA or a bioderived compound willinclude culturing a non-naturally occurring acetyl-CoA or a bioderivedcompound producing organism of the invention in sufficient nutrients andmedium to sustain and/or nearly sustain growth in an exponential phase.Continuous culture under such conditions can include, for example,growth or culturing for 1 day, 2, 3, 4, 5, 6 or 7 days or more.Additionally, continuous culture can include longer time periods of 1week, 2, 3, 4 or 5 or more weeks and up to several months.Alternatively, organisms of the invention can be cultured for hours, ifsuitable for a particular application. It is to be understood that thecontinuous and/or near-continuous culture conditions also can includeall time intervals in between these exemplary periods. It is furtherunderstood that the time of culturing the microbial organism of theinvention is for a sufficient period of time to produce a sufficientamount of product for a desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of acetyl-CoA or a bioderived compoundcan be utilized in, for example, fed-batch fermentation and batchseparation; fed-batch fermentation and continuous separation, orcontinuous fermentation and continuous separation. Examples of batch andcontinuous fermentation procedures are well known in the art.

In addition to the above fermentation procedures using the acetyl-CoA orthe bioderived compound producers of the invention for continuousproduction of substantial quantities of acetyl-CoA or a bioderivedcompound, the acetyl-CoA or the bioderived compound producers also canbe, for example, simultaneously subjected to chemical synthesis and/orenzymatic procedures to convert the product to other compounds or theproduct can be separated from the fermentation culture and sequentiallysubjected to chemical and/or enzymatic conversion to convert the productto other compounds, if desired.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of acetyl-CoA or abioderived compound.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Biirgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion or disruption strategies that result ingenetically stable microorganisms which overproduce the target product.Specifically, the framework examines the complete metabolic and/orbiochemical network of a microorganism in order to suggest geneticmanipulations that force the desired biochemical to become an obligatorybyproduct of cell growth. By coupling biochemical production with cellgrowth through strategically placed gene deletions or other functionalgene disruption, the growth selection pressures imposed on theengineered strains after long periods of time in a bioreactor lead toimprovements in performance as a result of the compulsory growth-coupledbiochemical production. Lastly, when gene deletions are constructedthere is a negligible possibility of the designed strains reverting totheir wild-type states because the genes selected by OptKnock are to becompletely removed from the genome. Therefore, this computationalmethodology can be used to either identify alternative pathways thatlead to biosynthesis of a desired product or used in connection with thenon-naturally occurring microbial organisms for further optimization ofbiosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat allow an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. publication2009/0047719, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

As disclosed herein, a nucleic acid encoding a desired activity of anacetyl-CoA or a bioderived compound pathway can be introduced into ahost organism. In some cases, it can be desirable to modify an activityof an acetyl-CoA or a bioderived compound pathway enzyme or protein toincrease production of acetyl-CoA or a bioderived compound. For example,known mutations that increase the activity of a protein or enzyme can beintroduced into an encoding nucleic acid molecule. Additionally,optimization methods can be applied to increase the activity of anenzyme or protein and/or decrease an inhibitory activity, for example,decrease the activity of a negative regulator.

One such optimization method is directed evolution. Directed evolutionis a powerful approach that involves the introduction of mutationstargeted to a specific gene in order to improve and/or alter theproperties of an enzyme. Improved and/or altered enzymes can beidentified through the development and implementation of sensitivehigh-throughput screening assays that allow the automated screening ofmany enzyme variants (for example, >10⁴). Iterative rounds ofmutagenesis and screening typically are performed to afford an enzymewith optimized properties. Computational algorithms that can help toidentify areas of the gene for mutagenesis also have been developed andcan significantly reduce the number of enzyme variants that need to begenerated and screened. Numerous directed evolution technologies havebeen developed (for reviews, see Hibbert et al., Biomol. Eng 22:11-19(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical andbiotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press;Otten and Quax. Biomol. Eng 22:1-9 (2005); and Sen et al., Appl Biochem.Biotechnol 143:212-223 (2007)) to be effective at creating diversevariant libraries, and these methods have been successfully applied tothe improvement of a wide range of properties across many enzymeclasses. Enzyme characteristics that have been improved and/or alteredby directed evolution technologies include, for example:selectivity/specificity, for conversion of non-natural substrates,temperature stability, for robust high temperature processing; pHstability, for bioprocessing under lower or higher pH conditions;substrate or product tolerance, so that high product titers can beachieved; binding (K_(m)), including broadening substrate binding toinclude non-natural substrates; inhibition (K_(i)), to remove inhibitionby products, substrates, or key intermediates; activity (kcat), toincreases enzymatic reaction rates to achieve desired flux; expressionlevels, to increase protein yields and overall pathway flux; oxygenstability, for operation of air sensitive enzymes under aerobicconditions; and anaerobic activity, for operation of an aerobic enzymein the absence of oxygen.

A number of exemplary methods have been developed for the mutagenesisand diversification of genes to target desired properties of specificenzymes. Such methods are well known to those skilled in the art. Any ofthese can be used to alter and/or optimize the activity of an acetyl-CoAor a bioderived compound pathway enzyme or protein. Such methodsinclude, but are not limited to EpPCR, which introduces random pointmutations by reducing the fidelity of DNA polymerase in PCR reactions(Pritchard et al., J. Theor. Biol. 234:497-509 (2005)); Error-proneRolling Circle Amplification (epRCA), which is similar to epPCR except awhole circular plasmid is used as the template and random 6-mers withexonuclease resistant thiophosphate linkages on the last 2 nucleotidesare used to amplify the plasmid followed by transformation into cells inwhich the plasmid is re-circularized at tandem repeats (Fujii et al.,Nucleic Acids Res. 32:e145 (2004); and Fujii et al., Nat. Protoc.1:2493-2497 (2006)); DNA or Family Shuffling, which typically involvesdigestion of two or more variant genes with nucleases such as Dnase I orEndoV to generate a pool of random fragments that are reassembled bycycles of annealing and extension in the presence of DNA polymerase tocreate a library of chimeric genes (Stemmer, Proc Natl Acad Sci USA91:10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994));Staggered Extension (StEP), which entails template priming followed byrepeated cycles of 2 step PCR with denaturation and very short durationof annealing/extension (as short as 5 sec) (Zhao et al., Nat.Biotechnol. 16:258-261 (1998)); Random Priming Recombination (RPR), inwhich random sequence primers are used to generate many short DNAfragments complementary to different segments of the template (Shao etal., Nucleic Acids Res 26:681-683 (1998)).

Additional methods include Heteroduplex Recombination, in whichlinearized plasmid DNA is used to form heteroduplexes that are repairedby mismatch repair (Volkov et al, Nucleic Acids Res. 27:e18 (1999); andVolkov et al., Methods Enzymol. 328:456-463 (2000)); RandomChimeragenesis on Transient Templates (RACHITT), which employs Dnase Ifragmentation and size fractionation of single stranded DNA (ssDNA) (Cowet al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extension onTruncated templates (RETT), which entails template switching ofunidirectionally growing strands from primers in the presence ofunidirectional ssDNA fragments used as a pool of templates (Lee et al.,J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide GeneShuffling (DOGS), in which degenerate primers are used to controlrecombination between molecules; (Bergquist and Gibbs, Methods Mol. Biol352:191-204 (2007); Bergquist et al., Biomol. Eng 22:63-72 (2005); Gibbset al., Gene 271:13-20 (2001)); Incremental Truncation for the Creationof Hybrid Enzymes (ITCHY), which creates a combinatorial library with 1base pair deletions of a gene or gene fragment of interest (Ostermeieret al., Proc. Natl. Acad. Sci. USA 96:3562-3567 (1999); and Ostermeieret al., Nat. Biotechnol. 17:1205-1209 (1999)); Thio-IncrementalTruncation for the Creation of Hybrid Enzymes (THIO-ITCHY), which issimilar to ITCHY except that phosphothioate dNTPs are used to generatetruncations (Lutz et al., Nucleic Acids Res 29:H16 (2001)); SCRATCHY,which combines two methods for recombining genes, ITCHY and DNAshuffling (Lutz et al., Proc. Natl. Acad. Sci. USA 98:11248-11253(2001)); Random Drift Mutagenesis (RNDM), in which mutations made viaepPCR are followed by screening/selection for those retaining usableactivity (Bergquist et al., Biomol. Eng. 22:63-72 (2005)); SequenceSaturation Mutagenesis (SeSaM), a random mutagenesis method thatgenerates a pool of random length fragments using random incorporationof a phosphothioate nucleotide and cleavage, which is used as a templateto extend in the presence of “universal” bases such as inosine, andreplication of an inosine-containing complement gives random baseincorporation and, consequently, mutagenesis (Wong et al., Biotechnol.J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); andWong et al., Anal. Biochem. 341:187-189 (2005)); Synthetic Shuffling,which uses overlapping oligonucleotides designed to encode “all geneticdiversity in targets” and allows a very high diversity for the shuffledprogeny (Ness et al., Nat. Biotechnol. 20:1251-1255 (2002)); NucleotideExchange and Excision Technology NexT, which exploits a combination ofdUTP incorporation followed by treatment with uracil DNA glycosylase andthen piperidine to perform endpoint DNA fragmentation (Muller et al.,Nucleic Acids Res. 33:e117 (2005)).

Further methods include Sequence Homology-Independent ProteinRecombination (SHIPREC), in which a linker is used to facilitate fusionbetween two distantly related or unrelated genes, and a range ofchimeras is generated between the two genes, resulting in libraries ofsingle-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460(2001)); Gene Site Saturation Mutagenesis™ (GSSM™), in which thestarting materials include a supercoiled double stranded DNA (dsDNA)plasmid containing an insert and two primers which are degenerate at thedesired site of mutations (Kretz et al., Methods Enzymol. 388:3-11(2004)); Combinatorial Cassette Mutagenesis (CCM), which involves theuse of short oligonucleotide cassettes to replace limited regions with alarge number of possible amino acid sequence alterations (Reidhaar-Olsonet al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al.Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis(CMCM), which is essentially similar to CCM and uses epPCR at highmutation rate to identify hot spots and hot regions and then extensionby CMCM to cover a defined region of protein sequence space (Reetz etal., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the MutatorStrains technique, in which conditional is mutator plasmids, utilizingthe mutD5 gene, which encodes a mutant subunit of DNA polymerase III, toallow increases of 20 to 4000-X in random and natural mutation frequencyduring selection and block accumulation of deleterious mutations whenselection is not required (Selifonova et al., Appl. Environ. Microbiol.67:3645-3649 (2001)); Low et al., J. Mol. Biol. 260:359-3680 (1996)).

Additional exemplary methods include Look-Through Mutagenesis (LTM),which is a multidimensional mutagenesis method that assesses andoptimizes combinatorial mutations of selected amino acids (Rajpal etal., Proc. Natl. Acad. Sci. USA 102:8466-8471 (2005)); Gene Reassembly,which is a DNA shuffling method that can be applied to multiple genes atone time or to create a large library of chimeras (multiple mutations)of a single gene (Tunable GeneReassembly™ (TGR™) Technology supplied byVerenium Corporation), in Silico Protein Design Automation (PDA), whichis an optimization algorithm that anchors the structurally definedprotein backbone possessing a particular fold, and searches sequencespace for amino acid substitutions that can stabilize the fold andoverall protein energetics, and generally works most effectively onproteins with known three-dimensional structures (Hayes et al., Proc.Natl. Acad. Sci. USA 99:15926-15931 (2002)); and Iterative SaturationMutagenesis (ISM), which involves using knowledge of structure/functionto choose a likely site for enzyme improvement, performing saturationmutagenesis at chosen site using a mutagenesis method such as StratageneQuikChange (Stratagene; San Diego Calif.), screening/selecting fordesired properties, and, using improved clone(s), starting over atanother site and continue repeating until a desired activity is achieved(Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et al., Angew.Chem. Int. Ed Engl. 45:7745-7751 (2006)).

Any of the aforementioned methods for mutagenesis can be used alone orin any combination. Additionally, any one or combination of the directedevolution methods can be used in conjunction with adaptive evolutiontechniques, as described herein.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example I Formate Assimilation Pathways

This example describes enzymatic pathways for converting pyruvate toformaldehyde, and optionally in combination with producing acetyl-CoAand/or reproducing pyruvate.

Step E, FIG. 1: Formate Reductase

The conversion of formate to formaldehyde can be carried out by aformate reductase (step E, FIG. 1). A suitable enzyme for thesetransformations is the aryl-aldehyde dehydrogenase, or equivalently acarboxylic acid reductase, from Nocardia iowensis. Expression of the nptgene product improved activity of the enzyme via post-transcriptionalmodification. The npt gene encodes a specific phosphopantetheinetransferase (PPTase) that converts the inactive apo-enzyme to the activeholo-enzyme. Information related to these proteins and genes is shownbelow.

Protein GenBank ID GI number Organism Car AAR91681.1 40796035 Nocardiaiowensis (sp. NRRL 5646) Npt ABI83656.1 114848891 Nocardia iowensis (sp.NRRL 5646)

Additional car and npt genes can be identified based on sequencehomology.

Protein GenBank ID GI number Organism fadD9 YP_978699.1 121638475Mycobacterium bovis BCG BCG_2812c YP_978898.1 121638674 Mycobacteriumbovis BCG nfa20150 YP_118225.1 54023983 Nocardia farcinica IFM 10152nfa40540 YP_120266.1 54026024 Nocardia farcinica IFM 10152 SGR_6790YP_001828302.1 182440583 Streptomyces griseus subsp. griseus NBRC 13350SGR_665 YP_001822177.1 182434458 Streptomyces griseus subsp. griseusNBRC 13350 MSMEG_2956 YP_887275.1 118473501 Mycobacterium smegmatis MC2155 MSMEG 5739 YP_889972.1 118469671 Mycobacterium smegmatis MC2 155MSMEG_2648 YP_886985.1 118471293 Mycobacterium smegmatis MC2 155MAP1040c NP_959974.1 41407138 Mycobacterium avium subsp.paratuberculosis K-10 MAP2899c NP_961833.1 41408997 Mycobacterium aviumsubsp. paratuberculosis K-10 MMAR_2117 YP_001850422.1 183982131Mycobacterium marinumM MMAR 2936 YP_001851230.1 183982939 MycobacteriummarinumM MMAR_1916 YP_001850220.1 183981929 Mycobacterium marinumMTpauDRAFT_33060 ZP_04027864.1 227980601 Tsukamurella paurometabola DSM20162 TpauDRAFT_20920 ZP_04026660.1 227979396 Tsukamurella paurometabolaDSM 20162 CPCC70011320 ZP_05045132.1 254431429 Cyanobium PCC7001DDBDRAFT_0187729 XP_636931.1 66806417 Dictyostelium discoideum AX4

An additional enzyme candidate found in Streptomyces griseus is encodedby the griC and griD genes. This enzyme is believed to convert3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzaldehyde.Co-expression of griC and griD with SGR 665, an enzyme similar insequence to the Nocardia iowensis npt, can be beneficial. Informationrelated to these proteins and genes is shown below.

Protein GenBank ID GI number Organism griC YP_001825755.1 182438036Streptomycesgriseus subsp. griseus NBRC 13350 griD YP_001825756.1182438037 Streptomycesgriseus subsp. griseus NBRC 13350

An enzyme with similar characteristics, alpha-aminoadipate reductase(AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in somefungal species. This enzyme naturally reduces alpha-aminoadipate toalpha-aminoadipate semialdehyde. The carboxyl group is first activatedthrough the ATP-dependent formation of an adenylate that is then reducedby NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizesmagnesium and requires activation by a PPTase. Enzyme candidates for AARand its corresponding PPTase are shown below.

Protein Gen Bank ID GI number Organism LYS2 AAA34747.1 171867Saccharomyces cerevisiae LYSS P50113.1 1708896 Saccharomyces cerevisiaeLYS2 AAC02241.1 2853226 Candida albicans LYSS AAO26020.1 28136195Candida albicans Lys1p P40976.3 13124791 Schizosaccharomyces pombe Lys7pQ10474.1 1723561 Schizosaccharomyces pombe Lys2 CAA74300.1 3282044Penicillium chrysogenum

Tani et al (Agric Biol Chem, 1978, 42: 63-68; Agric Biol Chem, 1974, 38:2057-2058) showed that purified enzymes from Escherichia coli strain Bcould reduce the sodium salts of different organic acids (e.g. formate,glycolate, acetate, etc.) to their respective aldehydes (e.g.formaldehyde, glycoaldehyde, acetaldehyde, etc.). Of three purifiedenzymes examined by Tani et al (1978), only the “A” isozyme was shown toreduce formate to formaldehyde. Collectively, this group of enzymes wasoriginally termed glycoaldehyde dehydrogenase; however, their novelreductase activity led the authors to propose the name glycolatereductase as being more appropriate (Morita et al, Agric Biol Chem,1979, 43: 185-186). Morita et al (Agric Biol Chem, 1979, 43: 185-186)subsequently showed that glycolate reductase activity is relativelywidespread among microorganisms, being found for example in:Pseudomonas, Agrobacterium, Escherichia, Flavobacterium, Micrococcus,Staphylococcus, Bacillus, and others. Without wishing to be bound by anyparticular theory, it is believed that some of these glycolate reductaseenzymes are able to reduce formate to formaldehyde.

Any of these CAR or CAR-like enzymes can exhibit formate reductaseactivity or can be engineered to do so.

Step F, FIG. 1 Formate Ligase, Formate Transferase, Formate Synthetase

The acylation of formate to formyl-CoA is catalyzed by enzymes withformate transferase, synthetase, or ligase activity (Step F, FIG. 1).Formate transferase enzymes have been identified in several organismsincluding Escherichia coli, Oxalobacter formigenes, and Lactobacillusacidophilus. Homologs exist in several other organisms. Enzymes actingon the CoA-donor for formate transferase may also be expressed to ensureefficient regeneration of the CoA-donor. For example, if oxalyl-CoA isthe CoA donor substrate for formate transferase, an additionaltransferase, synthetase, or ligase may be required to enable efficientregeneration of oxalyl-CoA from oxalate. Similarly, if succinyl-CoA oracetyl-CoA is the CoA donor substrate for formate transferase, anadditional transferase, synthetase, or ligase may be required to enableefficient regeneration of succinyl-CoA from succinate or acetyl-CoA fromacetate, respectively.

Protein Gen Bank ID GI number Organism YfdW NP_416875.1 16130306Escherichia coli frc O06644.3 21542067 Oxalobacter formiyenes frcZP_04021099.1 227903294 Lactobacillus acidophilus

Suitable CoA-donor regeneration or formate transferase enzymes areencoded by the gene products of cat1, cat2, and cat3 of Clostridiumkluyveri. These enzymes have been shown to exhibit succinyl-CoA,4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferase activity,respectively. Similar CoA transferase activities are also present inTrichomonas vaginalis and Trypanosoma brucei. Yet another transferasecapable of the desired conversions is butyryl-CoA:acetoacetateCoA-transferase. Exemplary enzymes are shown below. Genes FN0272 andFN0273 have been annotated as a butyrate-acetoacetate CoA-transferase

Protein GenBank ID GI number Organism Cat1 P38946.1 729048 Clostridiumkluyveri Cat2 P38942.2 1705614 Clostridium kluyveri Cat3 EDK35586.1146349050 Clostridium kluyveri TVAG_395550 XP_001330176 123975034Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875 Trypanosomabrucei FN0272 NP_603179.1 19703617 Fusobacterium nucleatum FN0273NP_603180.1 19703618 Fusobacterium nucleatum FN1857 NP_602657.1 19705162Fusobacterium nucleatum FN1856 NP_602656.1 19705161 Fusobacteriumnucleatum PG1066 NP_905281.1 34540802 Porphyromonas gingivalis W83PG1075 NP_905290.1 34540811 Porphyromonas gingivalis W83 TTE0720NP_622378.1 20807207 Thermoanaerobacter tengcongensis MB4 TTE0721NP_622379.1 20807208 Thermoanaerobacter tengcongensis MB4

Additional transferase enzymes of interest include proteins and genesshown below.

Protein GenBank ID GI number Organism AtoA P76459.1 2492994 Escherichiacoli AtoD P76458.1 2492990 Escherichia coli CtfA NP_149326.1 15004866Clostridium acetobutylicum CtfB NP_149327.1 15004867 Clostridiumacetobutylicum CtfA AAP42564.1 31075384 Clostridiumsaccharoperbutylacetonicum CtfB AAP42565.1 31075385 Clostridiumsaccharoperbutylacetonicum

Succinyl-CoA:3-ketoacid-CoA transferase naturally converts succinate tosuccinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid.Exemplary succinyl-CoA:3:ketoacid-CoA transferases proteins and genesare shown below.

Protein GenBank ID GI number Organism HPAG1 0676 YP_627417 108563101Helicobacter pylori HPAG1_0677 YP_627418 108563102 Helicobacter pyloriScoA NP_391778 16080950 Bacillus subtilis ScoB NP_391777 16080949Bacillus subtilis OXCT1 NP_000427 4557817 Homo sapiens OXCT2 NP_07140311545841 Homo sapiens

Two additional enzymes that catalyze the activation of formate toformyl-CoA reaction are AMP-forming formyl-CoA synthetase andADP-forming formyl-CoA synthetase. Exemplary enzymes, known to functionon acetate, are shown below. Such enzymes may also acylate formatenaturally or can be engineered to do so.

Protein GenBank ID GI Number Organism acs AAC77039.1 1790505 Escherichiacoli acoE AAA21945.1 141890 Ralstonia eutropha acs1 ABC87079.1 86169671Methanothermobacter thermautotrophicus acs1 AAL23099.1 16422835Salmonella enterica ACS1 Q01574.2 257050994 Saccharomyces cerevisiae

ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is anothercandidate enzyme that couples the conversion of acyl-CoA esters to theircorresponding acids with the concurrent synthesis of ATP. Severalenzymes with broad substrate specificities have been described in theliterature. Such enzymes may also acylate formate naturally or can beengineered to do so. Information related to these proteins and genes isshown below.

Protein GenBank ID GI number Organism AF1211 NP_070039.1 11498810Archaeoylobiis fulyidiis DSM 4304 AF1983 NP_070807.1 11499565Archaeoylobiis fulyidiis DSM 4304 scs YP_135572.1 55377722 Haloarculamarismortui ATCC 43049 PAE3250 NP_560604.1 18313937 Pyrobaculumaerophilum str. IM2 sucC NP_415256.1 16128703 Escherichia coli sucDAAC73823.1 1786949 Escherichia coli paaF AAC24333.2 22711873 Pseudomonasputida

An alternative method for adding the CoA moiety to formate is to apply apair of enzymes such as a phosphate-transferring acyltransferase and akinase. These activities enable the net formation of formyl-CoA with thesimultaneous consumption of ATP. An exemplary phosphate transferringacyltransferase is phosphotransacetylase, encoded by pta. Exemplaryenzymes are shown below. Such enzymes may also phosphorylate formatenaturally or can be engineered to do so.

Protein GenBank ID GI number Organism Pta NP_416800.1 16130232Escherichia coli Pta NP_461280.1 16765665 Salmonella enterica subsp.enterica serovar Typhimurium str. LT2 PAT2 XP_001694504.1 159472743Chlamydomonas reinhardtii PAT1 XP_001691787.1 159467202 Chlamydomonasreinhardtii

An exemplary acetate kinase is the E. coli acetate kinase, encoded byackA (Skarstedt and Silverstein J. Biol. Chem. 251:6775-6783 (1976)).Homologs exist in several other organisms including Salmonella entericaand Chlamydomonas reinhardtii. It is likely that such enzymes naturallypossess formate kinase activity or can be engineered to have thisactivity. Information related to these proteins and genes is shownbelow:

Protein GenBank ID GI number Organism AckA NP_416799.1 16130231Escherichia coli AckA NP_461279.1 16765664 Salmonella enterica subsp.enterica serovar Typhimurium str. LT2 ACK1 XP_001694505.1 159472745Chlamydomonas reinhardtii ACK2 XP_001691682.1 159466992 Chlamydomonasreinhardtii

The acylation of formate to formyl-CoA can also be carried out by aformate ligase. Such enzymes may also acylate formate naturally or canbe engineered to do so. Information related to these proteins and genesis shown below.

Protein GenBank ID GI number Organism SucC NP_415256.1 16128703Escherichia coli SucD AAC73823.1 1786949 Escherichia coli LSC1 NP_0147856324716 Saccharomyces cerevisiae LSC2 NP_011760 6321683 Saccharomycescerevisiae

Additional exemplary CoA-ligases include the rat dicarboxylate-CoAligase for which the sequence is yet uncharacterized (Vamecq et al.,Biochemical J. 230:683-693 (1985)), and exemplary enzymes shown below.Such enzymes may also acylate formate naturally or can be engineered todo so. Information related to these proteins and genes is shown below.

Protein GenBank ID GI number Organism Phl CAJ15517.1 77019264Penicillium chrysogenum PhlB ABS19624.1 152002983 Penicilliumchrysogenum PaaF AAC24333.2 22711873 Pseudomonas putida BioW NP_390902.250812281 Bacillus subtilis AACS NP_084486.1 21313520 Mus musculus AACSNP_076417.2 31982927 Homo sapiens Msed_1422 YP_001191504 146304188Metallosphaera sedula

Step G, FIG. 1: Formyl-CoA Reductase

Several acyl-CoA dehydrogenases are capable of reducing an acyl-CoA(e.g., formyl-CoA) to its corresponding aldehyde (e.g., formaldehyde)(Steps F, FIG. 1). Exemplary genes that encode such enzymes includethose below. Such enzymes may be capable of naturally convertingformyl-CoA to formaldehyde or can be engineered to do so.

Protein GenBank ID GI number Organism acr1 YP_047869.1 50086355Acinetobacter calcoaceticus acr1 AAC45217 1684886 Acinetobacter baylyiacr1 BAB85476.1 18857901 Acinetobacter sp. Strain M-1 sucD P38947.1172046062 Clostridium kluyveri sucD NP_904963.1 34540484 Porphyromonasgingivalis bphG BAA03892.1 425213 Pseudomonas sp adhE AAV66076.155818563 Leuconostoc mesenteroides Bld AAP42563.1 31075383 Clostridiumsaccharoperbutylacetonicum Ald ACL06658.1 218764192 Desulfatibacillumalkenivorans AK-01 Ald YP_001452373 157145054 Citrobacter koseri ATCCBAA-895 pduP NP_460996.1 16765381 Salmonella enterica Typhimurium pduPABJ64680.1 116099531 Lactobacillus brevis ATCC 367 BselDRAFT_1651ZP_02169447 163762382 Bacillus selenitireducens MLS10

An additional enzyme type that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase which transforms malonyl-CoA tomalonic semialdehyde. Malonyl-CoA reductase is a key enzyme inautotrophic carbon fixation via the 3-hydroxypropionate cycle inthermoacidophilic archaeal bacteria (Berg et al., Science 318:1782-1786(2007); Thauer, Science 318:1732-1733 (2007)). The enzyme utilizes NADPHas a cofactor. Although the aldehyde dehydrogenase functionality ofthese enzymes is similar to the bifunctional dehydrogenase fromChloroflexus aurantiacus, there is little sequence similarity. Bothmalonyl-CoA reductase enzyme candidates have high sequence similarity toaspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reductionand concurrent dephosphorylation of aspartyl-4-phosphate to aspartatesemialdehyde. Additional gene candidates can be found by sequencehomology to proteins in other organisms including Sulfolobussolfataricus and Sulfolobus acidocaldarius and have been listed below.Such enzymes may be capable of naturally converting formyl-CoA toformaldehyde or can be engineered to do so.

Protein GenBank ID GI number Organism Msed_0709 YP_001190808.1 146303492Metallosphaera sedula Mcr NP_378167.1 15922498 Sulfolobus tokodaii asd-2NP_343563.1 15898958 Sulfolobus solfataricus Saci 2370 YP_256941.170608071 Sulfolobus acidocaldarius Ald AAT66436 9473535 Clostridiumbeijerinckii eutE AAA80209 687645 Salmonella typhimurium eutE P774452498347 Escherichia coli

Step H, FIG. 1: Formyltetrahydrofolate Synthetase

Formyltetrahydrofolate synthetase ligates formate to tetrahydrofolate atthe expense of one ATP. This enzyme is found in several other organismsas listed below.

Protein GenBank ID GI number Organism Moth_0109 YP_428991.1 83588982Moorella thermoacetica CHY_2385 YP_361182.1 78045024 Carboxydothermushydrogenoformans FHS P13419.1 120562 Clostridium aciduriciCcarbDRAFT_1913 ZP_05391913.1 255524966 Clostridium carboxidivorans P7CcarbDRAFT_2946 ZP_05392946.1 255526022 Clostridium carboxidivorans P7Dhaf_0555 ACL18622.1 219536883 Desulfitobacterium hafniense fhsYP_001393842.1 153953077 Clostridium kluyveri DSM 555 fhs YP_003781893.1300856909 Clostridium ljungdahlii DSM 13528 MGA3_08300 EIJ83208.1387590889 Bacillus methanolicus MGA3 PB1_13509 ZP_10132113.1 387929436Bacillus methanolicus PB1

Steps I and J, FIG. 1: Formyltetrahydrofolate Synthetase andMethylenetetrahydrofolate Dehydrogenase

In M. thermoacetica, E coli, and C. hydrogenoformans,methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolatedehydrogenase are carried out by the bi-functional gene products.Several other organisms also encode for this bifunctional protein astabulated below.

Protein GenBank ID GI number Organism Moth_1516 YP_430368.1 83590359Moorella thermoacetica folD NP_415062.1 16128513 Escherichia coliCHY_1878 YP_360698.1 78044829 Carboxydothermus hydrogenoformansCcarbDRAFT_2948 ZP_05392948.1 255526024 Clostridium carboxidivorans P7folD ADK16789.1 300437022 Clostridium ljungdahlii DSM 13528 folD-2NP_951919.1 39995968 Geobacter sulfurreducens PCA folD YP_725874.1113867385 Ralstonia eutropha H16 folD NP_348702.1 15895353 Clostridiumacetobutylicum ATCC 824 folD YP_696506.1 110800457 Clostridiumperfringens MGA3_09460 EIJ83438.1 387591119 Bacillus methanolicus MGA3PB1_14689 ZP_10132349.1 387929672 Bacillus methanolicus PB1

Steps K, FIG. 1: Formaldehyde-Forming Enzyme or Spontaneous

Methylene-THF, or active formaldehyde, will spontaneously decompose toformaldehyde and THF. To achieve higher rates, a formaldehyde-formingenzyme can be applied. Such an activity can be obtained by engineeringan enzyme that reversibly forms methylene-THF from THF and aformaldehyde donor, to release flee formaldehyde. Such enzymes includeglycine cleavage system enzymes which naturally transfer a formaldehydegroup from methylene-THF to glycine (see Step L, FIG. 1 for candidateenzymes). Additional enzymes include serine hydroxymethyltransferase(see Step M, FIG. 1 for candidate enzymes), dimethylglycinedehydrogenase (Porter, et al., Arch Biochem Biophys. 1985, 243(2)396-407; Brizio et al., 2004, (37) 2, 434-442), sarcosine dehydrogenase(Porter, et al., Arch Biochem Biophys. 1985, 243(2) 396-407), anddimethylglycine oxidase (Leys, et al., 2003, The EMBO Journal 22(16)4038-4048).

Protein GenBank ID GI number Organism dmgo ZP_09278452.1 359775109Arthrobacter globiformis dmgo YP_002778684.1 226360906 Rhodococcusopacus B4 dmgo EFY87157.1 322695347 Metarhizium acridum CQMa 102 shdAAD53398.2 5902974 Homo sapiens shd NP_446116.1 GI: 25742657 Rattusnorvegicus dmgdh NP_037523.2 24797151 Homo sapiens dmgdh Q63342.12498527 Rattus norvegicus

Step L, FIG. 1: Glycine Cleavage System

The reversible NAD(P)H-dependent conversion of5,10-methylenetetrahydrofolate and CO₂ to glycine is catalyzed by theglycine cleavage complex, also called glycine cleavage system, composedof four protein components; P, H, T and L. The glycine cleavage complexis involved in glycine catabolism in organisms such as E. coli andglycine biosynthesis in eukaryotes (Kikuchi et al, Proc Jpn Acad Ser84:246 (2008)). The glycine cleavage system of E. coli is encoded byfour genes: gcvPHT and 1pdA (Okamura et al, Eur J Biochem 216:539-48(1993); Heil et al, Microbiol 148:2203-14 (2002)). Activity of theglycine cleavage system in the direction of glycine biosynthesis hasbeen demonstrated in vivo in Saccharomyces cerevisiae (Maaheimo et al,Eur J Biochem 268:2464-79 (2001)). The yeast GCV is encoded by GCV1,GCV2, GCV3 and LPD1.

Protein GenBank ID GI Number Organism gcvP AAC75941.1 1789269Escherichia coli gcvT AAC75943.1 1789272 Escherichia coli gcvHAAC75942.1 1789271 Escherichia coli lpdA AAC73227.1 1786307 Escherichiacoli GCV1 NP_010302.1 6320222 Saccharomyces cerevisiae GCV2 NP_013914.16323843 Saccharomyces cerevisiae GCV3 NP_009355.3 269970294Saccharomyces cerevisiae LPD1 NP_116635.1 14318501 Saccharomycescerevisiae

Step M, FIG. 1: Serine Hydroxymethyltransferase

Conversion of glycine to serine is catalyzed by serinehydroxymethyltransferase, also called glycine hydroxymethyltranferase.This enzyme reversibly converts glycine and5,10-methylenetetrahydrofolate to serine and THF. Serinemethyltransferase has several side reactions including the reversiblecleavage of 3-hydroxyacids to glycine and an aldehyde, and thehydrolysis of 5,10-methenyl-THF to 5-formyl-THF. Exemplary enzymes arelisted below.

Protein GenBank ID GI Number Organism glyA AAC75604.1 1788902Escherichia coli SHM1 NP_009822.2 37362622 Saccharomyces cerevisiae SHM2NP_013159.1 6323087 Saccharomyces cerevisiae glyA AAA64456.1 496116Methylobacterium extorquens glyA AAK60516.1 14334055 Corynebacteriumglutamicum

Step N, FIG. 1: Serine Deaminase

Serine can be deaminated to pyruvate by serine deaminase. Exemplaryenzymes are listed below.

Protein GenBank ID GI Number Organism sdaA YP_490075.1 388477887Escherichia coli sdaB YP_491005.1 388478813 Escherichia coli tdcGYP_491301.1 388479109 Escherichia coli tdcB YP_491307.1 388479115Escherichia coli sdaA YP_225930.1 62390528 Corynebacterium sp.

Step O, FIG. 1: Methylenetetrahydrofolate Reductase

In M. thermoacetica, this enzyme is oxygen-sensitive and contains aniron-sulfur cluster (Clark and Ljungdahl, J. Biol. Chem. 259:10845-10849(1984). This enzyme is encoded by metF in E. coli (Sheppard et al., J.Bacteriol. 181:718-725 (1999) and CHY_1233 in C. hydrogenoformans (Wu etal., PLoS Genet. 1:e65 (2005). The M. thermoacetica genes, and its C.hydrogenoformans counterpart, are located near the CODH/ACS genecluster, separated by putative hydrogenase and heterodisulfide reductasegenes. Some additional gene candidates found bioinformatically arelisted below. In Acetobacterium woodii metF is coupled to the Rnfcomplex through RnfC2 (Poehlein et al, PLoS One. 7:e33439). Homologs ofRnfC are found in other organisms by blast search. The Rnf complex isknown to be a reversible complex (Fuchs (2011) Annu Rev. Microbiol.65:631-658).

Protein GenBank ID GI number Organism Moth_1191 YP_430048.1 83590039Moorella thermoacetica Moth_1192 YP_430049.1 83590040 Moorellathermoacetica metF NP_418376.1 16131779 Escherichia coli CHY_1233YP_360071.1 78044792 Carboxydothermus hydrogenoformans CLJU_c37610YP_003781889.1 300856905 Clostridium ljungdahlii DSM 13528DesfrDRAFT_3717 ZP_07335241.1 303248996 Desulfovibrio fructosovorans JJCcarbDRAFT_2950 ZP_05392950.1 255526026 Clostridium carboxidivoransP7Ccel74_010100023124 ZP_07633513.1 307691067 Clostridium cellulovorans743B Cphy_3110 YP_001560205.1 160881237 Clostridium phytofermentans ISDg

Step P, FIG. 1: Acetyl-CoA Synthase

Acetyl-CoA synthase is the central enzyme of the carbonyl branch of theWood-Ljungdahl pathway. It catalyzes the synthesis of acetyl-CoA fromcarbon monoxide, coenzyme A, and the methyl group from a methylatedcorrinoid-iron-sulfur protein. The corrinoid-iron-sulfur-protein ismethylated by methyltetrahydrofolate via a methyltransferase. Expressionin a foreign host entails introducing one or more of the followingproteins and their corresponding activities:Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE),Corrinoid iron-sulfur protein (AcsD), Nickel-protein assembly protein(AcsF), Ferredoxin (Orf7), Acetyl-CoA synthase (AcsB and AcsC), Carbonmonoxide dehydrogenase (AcsA), and Nickel-protein assembly protein(CooC).

The genes used for carbon-monoxide dehydrogenase/acetyl-CoA synthaseactivity typically reside in a limited region of the native genome thatcan be an extended operon (Ragsdale, S W., Crit. Rev. Biochem. Mol.Biol. 39:165-195 (2004); Morton et at, J. Biol. Chem. 266:23824-23828(1991); Roberts et al., Proc. Natl. Acad. Sci. USA. 86:32-36 (1989).Each of the genes in this operon from the acetogen, M. thermoacetica,has already been cloned and expressed actively in E. coli (Morton et al.supra; Roberts et al. supra; Lu et al., J. Biol. Chem. 268:5605-5614(1993). The protein sequences of these genes can be identified by thefollowing GenBank accession numbers.

Protein GenBank ID GI number Organism AcsE YP_430054 83590045 Moorellathermoacetica AcsD YP_430055 83590046 Moorella thermoacetica AcsFYP_430056 83590047 Moorella thermoacetica Orf7 YP_430057 83590048Moorella thermoacetica AcsC YP_430058 83590049 Moorella thermoaceticaAcsB YP_430059 83590050 Moorella thermoacetica AcsA YP_430060 83590051Moorella thermoacetica CooC YP_430061 83590052 Moorella thermoacetica

The hydrogenic bacterium, Carboxydothermus hydrogenoformans, can utilizecarbon monoxide as a growth substrate by means of acetyl-CoA synthase(Wu et al., PLoS Genet. 1:e65 (2005)). In strain Z-2901, the acetyl-CoAsynthase enzyme complex lacks carbon monoxide dehydrogenase due to aframeshift mutation (Wu et al. supra (2005)), whereas in strain DSM6008, a functional unframeshifted full-length version of this proteinhas been purified (Svetlitchnyi et al., Proc. Natl. Acad. Sci. USA.101:446-451 (2004)). The protein sequences of the C. hydrogenoformansgenes from strain Z-2901 can be identified by the following GenBankaccession numbers.

Protein GenBank ID GI number Organism AcsE YP_360065 78044202Carboxydothermus hydrogenoformans AcsD YP_360064 78042962Carboxydothermus hydrogenoformans AcsF YP_360063 78044060Carboxydothermus hydrogenoformans Orf7 YP_360062 78044449Carboxydothermus hydrogenoformans AcsC YP_360061 78043584Carboxydothermus hydrogenoformans AcsB YP_360060 78042742Carboxydothermus hydrogenoformans CooC YP_360059 78044249Carboxydothermus hydrogenoformans

Homologous ACS/CODH genes can also be found in the draft genome assemblyof Clostridium carboxidivorans P7.

Protein GenBank ID GI Number Organism AcsA ZP_05392944.1 255526020Clostridium carboxidivorans P7 CooC ZP_05392945.1 255526021 Clostridiumcarboxidivorans P7 AcsF ZP_05392952.1 255526028 Clostridiumcarboxidivorans P7 AcsD ZP_05392953.1 255526029 Clostridiumcarboxidivorans P7 AcsC ZP_05392954.1 255526030 Clostridiumcarboxidivorans P7 AcsE ZP_05392955.1 255526031 Clostridiumcarboxidivorans P7 AcsB ZP_05392956.1 255526032 Clostridiumcarboxidivorans P7 Orf7 ZP_05392958.1 255526034 Clostridiumcarboxidivorans P7

The methanogenic archaeon, Methanosarcina acetivorans, can also grow oncarbon monoxide, exhibits acetyl-CoA synthase/carbon monoxidedehydrogenase activity, and produces both acetate and formate (Lessneret al., Proc. Natl. Acad. Sci. USA. 103:17921-17926 (2006)). Thisorganism contains two sets of genes that encode ACS/CODH activity(Rother and Metcalf, Proc. Natl. Acad Sci. USA. 101:16929-16934 (2004)).The protein sequences of both sets of M. acetivorans genes areidentified by the following GenBank accession numbers.

Protein GenBank ID GI number Organism AcsC NP_618736 20092661Methanosarcina acetivorans AcsD NP_618735 20092660 Methanosarcinaacetivorans AcsF, CooC NP_618734 20092659 Methanosarcina acetivoransAcsB NP_618733 20092658 Methanosarcina acetivorans AcsEps NP_61873220092657 Methanosarcina acetivorans AcsA NP_618731 20092656Methanosarcina acetivorans AcsC NP_615961 20089886 Methanosarcinaacetivorans AcsD NP_615962 20089887 Methanosarcina acetivorans AcsF,CooC NP_615963 20089888 Methanosarcina acetivorans AcsB NP_61596420089889 Methanosarcina acetivorans AcsEps NP_615965 20089890Methanosarcina acetivorans AcsA NP_615966 20089891 Methanosarcinaacetivorans

The AcsC, AcsD, AcsB, AcsEps, and AcsA proteins are commonly referred toas the gamma, delta, beta, epsilon, and alpha subunits of themethanogenic CODH/ACS. Homologs to the epsilon encoding genes are notpresent in acetogens such as M. thermoacetica or hydrogenogenic bacteriasuch as C. hydrogenoformans. Hypotheses for the existence of two activeCODH/ACS operons in M. acetivorans include catalytic properties (i.e.,K_(m), V_(max), k_(cat)) that favor carboxidotrophic or aceticlasticgrowth or differential gene regulation enabling various stimuli toinduce CODH/ACS expression (Rother et al., Arch. Microbiol. 188:463-472(2007)).

Step Y, FIG. 1: Glyceraldehydes-3-Phosphate Dehydrogenase and Enzymes ofLower Glycolysis

Enzymes comprising Step Y, G3P to PYR include:Glyceraldehyde-3-phosphate dehydrogenase; Phosphoglycerate kinase;Phosphoglyceromutase; Enolase; Pyruvate kinase or PTS-dependentsubstrate import.

Glyceraldehyde-3-phosphate dehydrogenase enzymes include: NADP-dependentglyceraldehyde-3-phosphate dehydrogenase, exemplary enzymes are:

Protein GenBank ID GI Number Organism gapN AAA91091.1 642667Streptococcus mutans NP-GAPDH AEC07555.1 330252461 Arabidopsis thalianaGAPN AAM77679.2 82469904 Triticum aestivum gapN CAI56300.1 87298962Clostridium acetobutylicum NADP-GAPDH 2D2I_A 112490271 Synechococcuselongatus PCC 7942 NADP-GAPDH CAA62619.1 4741714 Synechococcus elongatusPCC 7942 GDP1 XP_455496.1 50310947 Kluyveromyces lactis NRRL Y-1140HP1346 NP_208138.1 15645959 Helicobacter pylori 26695and NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, exemplaryenzymes are:

Protein GenBank ID GI Number Organism TDH1 NP_012483.1 6322409Saccharomyces cerevisiae s288c TDH2 NP_012542.1 6322468 Saccharomycescerevisiae s288c TDH3 NP_011708.1 632163 Saccharomyces cerevisiae s288cKLLA0A11858g XP_451516.1 50303157 Kluyveromyces lactis NRRL Y-1140KLLA0F20988g XP_456022.1 50311981 Kluyveromyces lactis NRRL Y-1140ANI_1_256144 XP_001397496.1 145251966 Aspergillus niger CBS 513.88YALI0C06369g XP_501515.1 50548091 Yarrowia lipolytica CTRG_05666XP_002551368.1 255732890 Candida tropicalis MYA-3404 HPODL 1089EFW97311.1 320583095 Hansenula polymorpha DL-1 gapA YP_490040.1388477852 Escherichia coli

Phosphoglycerate kinase enzymes include:

Protein GenBank ID GI Number Organism PGK1 NP_009938.2 10383781Saccharomyces cerevisiae s288c PGK BAD83658.1 57157302 Candida boidiniiPGK EFW98395.1 320584184 Hansenula polymorpha DL-1 pgk EIJ77825.1387585500 Bacillus methanolicus MGA3 pgk YP_491126.1 388478934Escherichia coli

Phosphoglyceromutase (aka phosphoglycerate mutase) enzymes include;

Protein GenBank ID GI Number Organism GPM1 NP_012770.1 6322697Saccharomyces cerevisiae s288c GPM2 NP_010263.1 6320183 Saccharomycescerevisiae s288c GPM3 NP_014585.1 6324516 Saccharomyces cerevisiae s288cHPODL 1391 EFW96681.1 320582464 Hansenula polymorpha DL-1 HPODL_0376EFW97746.1 320583533 Hansenula polymorpha DL-1 gpmI EIJ77827.1 387585502Bacillus methanolicus MGA3 gpmA YP_489028.1 388476840 Escherichia coligpmM AAC76636.1 1790041 Escherichia coli

Enolase (also known as phosphopyruvate hydratase and 2-phosphoglyceratedehydratase) enzymes include:

Protein GenBank ID GI Number Organism ENO1 NP_011770.3 398366315Saccharomyces cerevisiae s288c ENO2 AAB68019.1 458897 Saccharomycescerevisiae s288c HPODL 2596 EFW95743.1 320581523 Hansenula polymorphaDL-1 eno EIJ77828.1 387585503 Bacillus methanolicus MGA3 eno AAC75821.11789141 Escherichia coli

Pyruvate kinase (also known as phosphoenolpyruvate kinase andphosphoenolpyruvate kinase) or PTS-dependent substrate import enzymesinclude those below. Pyruvate kinase, also known as phosphoenolpyruvatesynthase (EC 2.7.9.2), converts pyruvate and ATP to PEP and AMP. Thisenzyme is encoded by the PYK1 (Burke et al., J. Biol. Chem.258:2193-2201 (1983)) and PYK2 (Boles et al., J. Bacteriol.179:2987-2993 (1997)) genes in S. cerevisiae. In E. coli, this activityis catalyzed by the gene products of pykF and pykA. Note that pykA andpykF are genes encoding separate enzymes potentially capable of carryingout the PYK reaction. Selected homologs of the S. cerevisiae enzymes arealso shown in the table below.

Protein GenBank ID GI Number Organism PYK1 NP_009362 6319279Saccharomyces cerevisiae PYK2 NP_014992 6324923 Saccharomyces cerevisiaepykF NP_416191.1 16129632 Escherichia coli pykA NP_416368.1 16129807Escherichia coli KLLA0F23397g XP_456122.1 50312181 Kluyveromyces lactisCaO19.3575 XP_714934.1 68482353 Candida albicans CaO19.11059 XP_714997.168482226 Candida albicans YALI0F09185p XP_505195 210075987 Yarrowialipolytica ANI_1_1126064 XP_001391973 145238652 Aspergillus nigerMGA3_03005 EIJ84220.1 387591903 Bacillus methanolicus MGA3 HPODL_1539EFW96829.1 320582612 Hansenula polymorpha DL-1

PTS-dependent substrate uptake systems catalyze a phosphotransfercascade that couples conversion of PEP to pyruvate with the transportand phosphorylation of carbon substrates. For example, the glucose PTSsystem transports glucose, releasing glucose-6-phosphate into thecytoplasm and concomitantly converting phosphoenolpyruvate to pyruvate.PTS systems are comprised of substrate-specific andnon-substrate-specific components. In E. coli the two non-specificcomponents are encoded by ptsI (Enzyme I) and ptsH (HPr). Thesugar-dependent components are encoded by crr and ptsG. Pts systems havebeen extensively studied and are reviewed, for example in Postma et al,Microbiol Rev 57: 543-94 (1993).

Protein GenBank ID GI Number Organism ptsG AC74185.1 1787343 Escherichiacoli ptsI AAC75469.1 1788756 Escherichia coli ptsH AAC75468.1 1788755Escherichia coli err AAC75470.1 1788757 Escherichia coli

The IIA[Glc] component mediates the transfer of the phosphoryl groupfrom histidine protein Hpr (ptsH) to the IIB[Glc] (ptsG) component. Atruncated variant of the crr gene was introduced into 1,4-butanediolproducing strains.

Alternatively, Phosphoenolpyruvate phosphatase (EC 3.1.3.60) catalyzesthe hydrolysis of PEP to pyruvate and phosphate. Numerous phosphataseenzymes catalyze this activity, including alkaline phosphatase (EC3.1.3.1), acid phosphatase (EC 3.1.3.2), phosphoglycerate phosphatase(EC 3.1.3.20) and PEP phosphatase (EC 3.1.3.60). PEP phosphatase enzymeshave been characterized in plants such as Vignia radiate, Bruguierasexangula and Brassica nigra. Exemplary enzymes are listed below. Enzymeengineering and/or removal of targeting sequences may be required foralkaline phosphatase enzymes to function in the cytoplasm.

Protein GenBank ID GI Number Organism phyA O00092.1 41017447 Aspergillusfumigatus Acp5 P13686.3 56757583 Homo sapiens phoA NP_414917.2 49176017Escherichia coli phoX ZP_01072054.1 86153851 Campylobacter jejuni PHO8AAA34871.1 172164 Saccharomyces cerevisiae SaurJH1_2706 YP_001317815.1150395140 Staphylococcus aureus

Step Q, FIG. 1: Pyruvate Formate Lyase

Pyruvate formate-lyase (PFL, EC 2.3.1.54), encoded by pflB in E. coli,can convert pyruvate into acetyl-CoA and formate. The activity of PFLcan be enhanced by an activating enzyme encoded by pflA. Keto-acidformate-lyase (EC 2.3.1.-), also known as 2-ketobutyrate formate-lyase(KFL) and pyruvate formate-lyase 4, is the gene product of tdcE in E.coli. This enzyme catalyzes the conversion of 2-ketobutyrate topropionyl-CoA and formate during anaerobic threonine degradation, andcan also substitute for pyruvate formate-lyase in anaerobic catabolism.The enzyme is oxygen-sensitive and, like PflB, can requirepost-translational modification by PFL-AE to activate a glycyl radicalin the active site (Hesslinger et al., Mol Microbiol 27:477-492 (1998)).Exemplary enzymes are listed below.

Protein GenBank ID GI Number Organism pflB NP_415423 16128870Escherichia coli pflA NP_415422.1 16128869 Escherichia coli tdcEAAT48170.1 48994926 Escherichia coli PflD NP_070278.1 11499044Archaeglubus fulgidus Pfl CAA03993 2407931 Lactococcus lactis PflBAA09085 1129082 Streptococcus mutans PFL1 XP_001689719.1 159462978Chlamydomonas reinhardtii pflA1 XP_001700657.1 159485246 Chlamydomonasreinhardtii Pfl Q46266.1 2500058 Clostridium pasteurianum Act CAA63749.11072362 Clostridium pasteurianum

Step R, FIG. 1: Pyruvate Dehydrogenase, Pyruvate FerredoxinOxidoreductase, Pyruvate:NADP+ Oxidoreductase

The pyruvate dehydrogenase (PDH) complex catalyzes the conversion ofpyruvate to acetyl-CoA (FIG. 3H). The E. coli PDH complex is encoded bythe genes aceEF and 1pdA. Enzyme engineering efforts have improved theE. coli PDH enzyme activity under anaerobic conditions (Kim et al., J.Bacteriol. 190:3851-3858 (2008); Kim et al., Appl. Environ. Microbiol.73:1766-1771 (2007); Zhou et al., Biotechnol. Lett. 30:335-342 (2008)).In contrast to the E. coli PDH, the B. subtilis complex is active andrequired for growth under anaerobic conditions (Nakano et al.,179:6749-6755 (1997)). The Klebsiella pneumoniae PDH, characterizedduring growth on glycerol, is also active under anaerobic conditions(Menzel et al., 56:135-142 (1997)). Comparative kinetics of Rattusnorvegicus PDH and BCKAD indicate that BCKAD has higher activity on2-oxobutanoate as a substrate (Paxton et al., Biochem J. 234:295-303(1986)). The S. cerevisiae PDH complex can consist of an E2 (LAT1) corethat binds E1 (PDA1, PDB1), E3 (LPD1), and Protein X (PDX1) components(Pronk et al., Yeast 12:1607-1633 (1996)). The PDH complex of S.cerevisiae is regulated by phosphorylation of E1 involving PKP1 (PDHkinase I), PTCS (PDH phosphatase I), PKP2 and PTC6. Modification ofthese regulators may also enhance PDH activity. Coexpression of lipoylligase (LplA of E. coli and AIM22 in S. cerevisiae) with PDH in thecytosol may be necessary for activating the PDH enzyme complex.Increasing the supply of cytosolic lipoate, either by modifying ametabolic pathway or media supplementation with lipoate, may alsoimprove PDH activity.

Gene Accession No. GI Number Organism aceE NP_414656.1 16128107Escherichia coli aceF NP_414657.1 16128108 Escherichia coli lpdNP_414658.1 16128109 Escherichia coli lplA NP_418803.1 16132203Escherichia coli pdhA P21881.1 3123238 Bacillus subtilis pdhB P21882.1129068 Bacillus subtilis pdhC P21883.2 129054 Bacillus subtilis pdhDP21880.1 118672 Bacillus subtilis aceE YP_001333808.1 152968699Klebsiella pneumoniae aceF YP_001333809.1 152968700 Klebsiellapneumoniae lpdA YP_001333810.1 152968701 Klebsiella pneumoniae Pdha1NP_001004072.2 124430510 Rattus norvegicus Pdha2 NP_446446.1 16758900Rattus norvegicus Dlat NP_112287.1 78365255 Rattus norvegicus DldNP_955417.1 40786469 Rattus norvegicus LAT1 NP_014328 6324258Saccharomyces cerevisiae PDA1 NP_011105 37362644 Saccharomycescerevisiae PDB1 NP_009780 6319698 Saccharomyces cerevisiae LPD1NP_116635 14318501 Saccharomyces cerevisiae PDX1 NP_011709 6321632Saccharomyces cerevisiae AIM22 NP_012489.2 83578101 Saccharomycescerevisiae

As an alternative to the large multienzyme PDH complexes describedabove, some organisms utilize enzymes in the 2-ketoacid oxidoreductasefamily (OFOR) to catalyze acylating oxidative decarboxylation of2-keto-acids. Unlike the PDH complexes, PFOR enzymes contain iron-sulfurclusters, utilize different cofactors and use ferredoxin or flavodixinas electron acceptors in lieu of NAD(P)H. Pyruvate ferredoxinoxidoreductase (PFOR) can catalyze the oxidation of pyruvate to formacetyl-CoA (FIG. 3H). Several additional PFOR enzymes are described inRagsdale, Chem. Rev. 103:2333-2346 (2003). Finally, flavodoxinreductases (e.g., fqrB from Helicobacter pylori or Campylobacter jejuni(St Maurice et al., J. Bacteriol. 189:4764-4773 (2007))) or Rnf-typeproteins (Seedorf et al., Proc. Natl. Acad. Sci. USA. 105:2128-2133(2008); Hellmann et al., J Bacteriol. 190:784-791 (2008)) provide ameans to generate NADH or NADPH from the reduced ferredoxin generated byPFOR These proteins are identified below.

Protein GenBank ID GI Number Organism Por CAA70873.1 1770208Desulfovibrio africanus Por YP_428946.1 83588937 Moorella thermoaceticaydbK NP_415896.1 16129339 Escherichia coli fqrB NP_207955.1 15645778Helicobacter pylori fqrB YP_001482096.1 157414840 Campylobacter jejuniRnfC EDK33306.1 146346770 Clostridium kluyveri RnfD EDK33307.1 146346771Clostridium kluyveri RnfG EDK33308.1 146346772 Clostridium kluyveri RnfEEDK33309.1 146346773 Clostridium kluyveri RnfA EDK33310.1 146346774Clostridium kluyveri RnfB EDK33311.1 146346775 Clostridium kluyveri

Pyruvate:NADP oxidoreductase (PNO) catalyzes the conversion of pyruvateto acetyl-CoA. This enzyme is encoded by a single gene and the activeenzyme is a homodimer, in contrast to the multi-subunit PDH enzymecomplexes described above. The enzyme from Euglena gracilis isstabilized by its cofactor, thiamin pyrophosphate (Nakazawa et al, ArchBiochem Biophys 411:183-8 (2003)). The mitochondrial targeting sequenceof this enzyme should be removed for expression in the cytosol. The PNOprotein of E gracilis and other NADP-dependent pyruvate:NADP+oxidoreductase enzymes are listed in the table below.

Protein GenBank ID GI Number Organism PNO Q94IN5.1 33112418 Euglenagracilis cgd4_690 XP_625673.1 66356990 Cryptosporidium parvumIowa IITPP_PFOR_PNO XP_002765111.11 294867463 Perkinsus marinus ATCC 50983

Step S, FIG. 1: Formate Dehydrogenase

Formate dehydrogenase (FDH) catalyzes the reversible transfer ofelectrons from formate to an acceptor. Enzymes with FDH activity utilizevarious electron Gathers such as, for example, NADH (EC 1.2.1.2), NADPH(EC 1.2.1.43), quinols (EC 1.1.5.6), cytochromes (EC 1.2.2.3) andhydrogenases (EC 1.1.99.33). The loci, Moth_2312 is responsible forencoding the alpha subunit of formate dehydrogenase while the betasubunit is encoded by Moth_2314. Another set of genes encoding formatedehydrogenase activity with a propensity for CO₂ reduction is encoded bySfum_2703 through Sfum_2706 in Syntrophobacter fumaroxidans (de Bok etal., Eur J Biochem. 270:2476-2485 (2003)); Reda et al., PNAS105:10654-10658 (2008)). A similar set of genes presumed to carry outthe same function are encoded by CHY_0731, CHY_0732, and CHY_0733 in C.hydrogenoformans (Wu et al., PLoS Genet 1:e65 (2005)).

Several EM8 enzymes have been identified that have higher specificityfor NADP as the cofactor as compared to NAD. This enzyme has been deemedas the NADP-dependent formate dehydrogenase and has been reported from 5species of the Burkholderia cepacia complex. More gene candidates can beidentified using sequence homology of proteins deposited in Publicdatabases such as NCBI, JGI and the metagenomic databases.

Protein GenBank ID GI Number Organism Moth_2312 YP_431142 148283121Moorella thermoacetica Moth_2314 YP_431144 83591135 Moorellathermoacetica Sfum 2703 YP_846816.1 116750129 Syntrophobacterfiimaroxidans Sfum 2704 YP_846817.1 116750130 Syntrophobacterfiimaroxidans Sfum_2705 YP_846818.1 116750131 Syntrophobacterfiimaroxidans Sfum_2706 YP_846819.1 116750132 Syntrophobacterfiimaroxidans CHY 0731 YP_359585.1 78044572 Carboxydothermushydrogenoformans CHY 0732 YP_359586.1 78044500 Carboxydothermushydrogenoformans CHY 0733 YP_359587.1 78044647 Carboxydothermushydrogenoformans CcarbDRAFT_0901 ZP_05390901.1 255523938 Clostridiumcarboxidivorans P7 CcarbDRAFT_4380 ZP_05394380.1 255527512 Clostridiumcarboxidivorans P7 fdhA, MGA3_06625 EIJ82879.1 387590560 Bacillusmethanolicus MGA3 fdhA, PB1_11719 ZP10131761.1 387929084 Bacillusmethanolicus PB1 fdhD, MGA3_06630 EIJ82880.1 387590561 Bacillusmethanolicus MGA3 fdhD, PB1_11724 ZP_10131762.1 387929085 Bacillusmethanolicus PB1 fdh ACF35003.1 194220249 Burkholderia stabilis fdhACF35004.1 194220251 Burkholderia pyrrocinia fdh ACF35002.1 194220247Burkholderia cenocepacia fdh ACF35001.1 194220245 Burkholderiamultivorans fdh ACF35000.1 194220243 Burkholderia cepacia FDH1AAC49766.1 2276465 Candida boidinii fdh CAA57036.1 1181204 Candidamethylica FDH2 P0CF35.1 294956522 Saccharomyces cerevisiae S288c FDH1NP_015033.1 6324964 Saccharomyces cerevisiae S288c fdsG YP_725156.1113866667 Ralstonia eutropha fdsB YP_725157.1 113866668 Ralstoniaeutropha fdsA YP_725158.1 113866669 Ralstonia eutropha fdsC YP_725159.1113866670 Ralstonia eutropha fdsD YP_725160.1 113866671 Ralstoniaeutropha

Example II Production of Reducing Equivalents

This example describes methanol metabolic pathways and other additionalenzymes generating reducing equivalents as shown in FIG. 2.

FIG. 2, Step A—Methanol Methyltransferase

A complex of 3-methyltransferase proteins, denoted MtaA, MtaB, and MtaC,perform the desired methanol methyltransferase activity (Ragsdale, S W.,Crit. Rev. Biochem. Mol. Biol. 39:165-195 (2004)).

MtaB is a zinc protein that can catalyze the transfer of a methyl groupfrom methanol to MtaC, a corrinoid protein. The protein sequences ofvarious MtaB and MtaC encoding genes in M. barkeri, M. acetivorans, andM. thermoaceticum can be identified by their following GenBank accessionnumbers.

Protein GenBank ID GI number Organism MtaB1 YP_304299 73668284Methanosarcina barkeri MtaC1 YP_304298 73668283 Methanosarcina barkeriMtaB2 YP_307082 73671067 Methanosarcina barkeri MtaC2 YP_307081 73671066Methanosarcina barkeri MtaB3 YP_304612 73668597 Methanosarcina barkeriMtaC3 YP_304611 73668596 Methanosarcina barkeri MtaB1 NP_615421 20089346Methanosarcina acetivorans MtaB1 NP_615422 20089347 Methanosarcinaacetivorans MtaB2 NP_619254 20093179 Methanosarcina acetivorans MtaC2NP_619253 20093178 Methanosarcina acetivorans MtaB3 NP_616549 20090474Methanosarcina acetivorans MtaC3 NP_616550 20090475 Methanosarcinaacetivorans MtaB YP_430066 83590057 Moorella thermoacetica MtaCYP_430065 83590056 Moorella thermoacetica MtaA YP_430064 83590056Moorella thermoacetica

In general, homology searches are an effective means of identifyingmethanol methyltransferases because MtaB encoding genes show little orno similarity to methyltransferases that act on alternative substratessuch as trimethylamine, dimethylamine, monomethylamine, ordimethylsulfide. Mutant strains lacking two of the sets were able togrow on methanol, whereas a strain lacking all three sets of MtaB andMtaC genes sets could not grow on methanol. This suggests that each setof genes plays a role in methanol utilization.

MtaA is zinc protein that catalyzes the transfer of the methyl groupfrom MtaC to either Coenzyme M in methanogens or methyltetrahydrofolatein acetogens. MtaA can also utilize methylcobalamin as the methyl donor.Exemplary genes encoding MtaA can be found in methanogenic archaea suchas Methanosarcina barkeri (Maeder et al., J. Bacteriol. 188:7922-7931(2006) and Methanosarcina acetivorans (Galagan et al., Genome Res.12:532-542 (2002), as well as the acetogen, Moorella thermoacetica ((Daset al., Proteins 67:167-176 (2007)). In general, MtaA proteins thatcatalyze the transfer of the methyl group from CH₃—MtaC are difficult toidentify bioinformatically as they share similarity to other corrinoidprotein methyltransferases and are not oriented adjacent to the MtaB andMtaC genes on the chromosomes. Nevertheless, a number of MtaA encodinggenes have been characterized. The protein sequences of these genes inM. barkeri and M. acetivorans can be identified by the following GenBankaccession numbers.

Protein GenBank ID GI number Organism MtaA YP_304602 73668587Methanosarcina barkeri MtaA1 NP_619241 20093166 Methanosarcinaacetivorans MtaA2 NP_616548 20090473 Methanosarcina acetivorans

The MtaA gene, YP_304602, from M. barkeri was cloned, sequenced, andfunctionally overexpressed in E. coli (Harms and Thauer, Eur. J.Biochem. 235:653-659 (1996)).

Putative MtaA encoding genes in M. thermoacetica were identified bytheir sequence similarity to the characterized methanogenic MtaA genes.Specifically, three M. thermoacetica genes show high homology (>30%sequence identity) to YP_304602 from M. barkeri. The protein sequencesof putative MtaA encoding genes from M. thermoacetica can be identifiedby the following GenBank accession numbers.

Protein GenBank ID GI number Organism MtaA YP_430937 83590928 Moorellathermoacetica MtaA YP_431175 83591166 Moorella thermoacetica MtaAYP_430935 83590926 Moorella thermoacetica MtaA YP_430064 83590056Moorella thermoacetica

FIG. 2, Step B—Methylenetetrahydrofolate Reductase

The conversion of methyl-THF to methylenetetrahydrofolate is catalyzedby methylenetetrahydrofolate reductase. Some additional gene candidatesfound bioinformatically are listed below. In Acetobacterium woodii metFis coupled to the Rnf complex through RnfC2 (Poehlein et al, PLoS One.7:e33439). Homologs of RnfC are found in other organisms by blastsearch. The Rnf complex is known to be a reversible complex (Fuchs(2011) Annu. Rev. Microbiol. 65:631-658).

Protein GenBank ID GI number Organism Moth_1191 YP_430048.1 83590039Moorella thermoacetica Moth_1192 YP_430049.1 83590040 Moorellathermoacetica metF NP_418376.1 16131779 Escherichia coli CHY_1233YP_360071.1 78044792 Carboxydothermus hydrogenoformans CLJU_c37610YP_003781889.1 300856905 Clostridium ljungdahlii DSM 13528DesfrDRAFT_3717 ZP_07335241.1 303248996 Desulfovibrio fructosovorans JJCcarbDRAFT_2950 ZP_05392950.1 255526026 Clostridium carboxidivorans P7Ccel74_010100023124 ZP_07633513.1 307691067 Clostridium cellulovorans743B Cphy_3110 YP_001560205.1 160881237 Clostridium phytofermentans ISDg

FIG. 2, Steps C and D—Methylenetetrahydrofolate Dehydrogenase,Methenyltetrahydrofolate Cyclohydrolase

In M. thermoacetica, E. coli, and C. hydrogenoformans,methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolatedehydrogenase are carried out by the bi-functional gene products ofMoth_1516, folD, and CHY 1878, respectively (Pierce et al., Environ.Microbiol. 10:2550-2573 (2008); Wu et al., PLoS Genet. 1:e65 (2005);D′Ari and Rabinowitz, J. Biol. Chem. 266:23953-23958 (1991)). A homologexists in C. carboxidivorans P7. Several other organisms also encode forthis bifunctional protein as tabulated below.

Protein GenBank ID GI number Organism Moth_1516 YP_430368.1 83590359Moorella thermoacetica folD NP_415062.1 16128513 Escherichia coliCHY_1878 YP_360698.1 78044829 Carboxydothermus hydrogenoformansCcarbDRAFT_2948 ZP_05392948.1 255526024 Clostridium carboxidivorans P7folD ADK16789.1 300437022 Clostridium ljungdahlii DSM 13528 folD-2NP_951919.1 39995968 Geobacter sulfurreducens PCA folD YP_725874.1113867385 Ralstonia eutropha H16 folD NP_348702.1 15895353 Clostridiumacetobutylicum ATCC 824 folD YP_696506.1 110800457 Clostridiumperfringens MGA3_09460 EIJ83438.1 387591119 Bacillus methanolicus MGA3PB1_14689 ZP_10132349.1 387929672 Bacillus methanolicus PB1

FIG. 2, Step E—Formyltetrahydrofolate Deformylase

This enzyme catalyzes the hydrolysis of 10-formyltetrahydrofolate(formyl-THF) to THF and formate. In E coli, this enzyme is encoded bypurU and has been overproduced, purified, and characterized (Nagy, etal., J. Bacteriol. 3:1292-1298 (1995)).

Protein GenBank ID GI number Organism purU AAC74314.1 1787483Escherichia coli K-12 MG1655 purU BAD97821.1 63002616 Corynebacteriumsp. U-96 purU EHE84645.1 354511740 Corynebacterium glutamicum ATCC 14067purU NP_460715.1 16765100 Salmonella enterica subsp. enterica serovarTyphimurium str. LT2

FIG. 2, Step F—Formyltetrahydrofolate Synthetase

Formyltetrahydrofolate synthetase ligates formate to tetrahydrofolate atthe expense of one ATP. This enzyme is found in several organisms aslisted below.

Protein GenBank ID GI number Organism Moth_0109 YP_428991.1 83588982Moorella thermoacetica CHY_2385 YP_361182.1 78045024 Carboxydothermushydrogenoformans FHS P13419.1 120562 Clostridium aciduriciCcarbDRAFT_1913 ZP_05391913.1 255524966 Clostridium carboxidivorans P7CcarbDRAFT_2946 ZP_05392946.1 255526022 Clostridium carboxidivorans P7Dhaf_0555 ACL18622.1 219536883 Desulfitobacterium hafniense fhsYP_001393842.1 153953077 Clostridium kluyveri DSM 555 fhs YP_003781893.1300856909 Clostridium ljungdahlii DSM 13528 MGA3_08300 EIJ83208.1387590889 Bacillus methanolicus MGA3 PB1_13509 ZP_10132113.1 387929436Bacillus methanolicus PB1

FIG. 2, Step G—Formate Hydrogen Lyase

A formate hydrogen lyase enzyme can be employed to convert formate tocarbon dioxide and hydrogen. An exemplary formate hydrogen lyase enzymecan be found in Escherichia coli. The E. coli formate hydrogen lyaseconsists of hydrogenase 3 and formate dehydrogenase-H (Maeda et al.,Appl Microbiol Biotechnol 77:879-890 (2007)). It is activated by thegene product of fhlA. (Maeda et al., Appl Microbiol Biotechnol77:879-890 (2007)). The addition of the trace elements, selenium, nickeland molybdenum, to a fermentation broth has been shown to enhanceformate hydrogen lyase activity (Soini et al., Microb. Cell Fact. 7:26(2008)). Various hydrogenase 3, formate dehydrogenase andtranscriptional activator genes are shown below.

Protein GenBank ID GI number Organism hycA NP_417205 16130632Escherichia coli K-12 MG1655 hycB NP_417204 16130631 Escherichia coliK-12 MG1655 hycC NP_417203 16130630 Escherichia coli K-12 MG1655 hycDNP_417202 16130629 Escherichia coli K-12 MG1655 hycE NP_417201 16130628Escherichia coli K-12 MG1655 hycF NP_417200 16130627 Escherichia coliK-12 MG1655 hycG NP_417199 16130626 Escherichia coli K-12 MG1655 hycHNP_417198 16130625 Escherichia coli K-12 MG1655 hycI NP_417197 16130624Escherichia coli K-12 MG1655 fdhF NP_418503 16131905 Escherichia coliK-12 MG1655 fhlA NP_417211 16130638 Escherichia coli K-12 MG1655

A formate hydrogen lyase enzyme also exists in the hyperthermophilicarchaeon, Thermococcus litoralis (Takacs et al., BMC Microbiol 8:88(2008)).

Protein GenBank ID GI number Organism mhyC ABW05543 157954626Thermococcus litoralis mhyD ABW05544 157954627 Thermococcus litoralismhyE ABW05545 157954628 Thermococcus litoralis myhF ABW05546 157954629Thermococcus litoralis myhG ABW05547 157954630 Thermococcus litoralismyhH ABW05548 157954631 Thermococcus litoralis fdhA AAB94932 2746736Thermococcus litoralis fdhB AAB94931 157954625 Thermococcus litoralis

Additional formate hydrogen lyase systems have been found in Salmonellatyphimurium, Klebsiella pneumoniae, Rhodospirillum rubrum,Methanobacterium formicicum (Vardar-Schara et al., MicrobialBiotechnology 1:107-125 (2008)).

FIG. 2, Step H—Hydrogenase

Hydrogenase enzymes can convert hydrogen gas to protons and transferelectrons to acceptors such as ferredoxins, NAD+, or NADP+. Ralstoniaeutropha H16 uses hydrogen as an energy source with oxygen as a terminalelectron acceptor. Its membrane-bound uptake [NiFe]-hydrogenase is an“02-tolerant” hydrogenase (Cracknell, et al. Proc Nat Acad Sci, 106(49)20681-20686 (2009)) that is periplasmically-oriented and connected tothe respiratory chain via a b-type cytochrome (Schink and Schlegel,Biochim. Biophys. Acta, 567, 315-324 (1979); Bernhard et al., Eur. JBiochem. 248, 179-186 (1997)). R. eutropha also contains an 02-tolerantsoluble hydrogenase encoded by the Hox operon which is cytoplasmic anddirectly reduces NAD+ at the expense of hydrogen (Schneider andSchlegel, Biochim. Biophys. Acta 452, 66-80 (1976); Burgdorf, J. Bact.187(9) 3122-3132 (2005)). Soluble hydrogenase enzymes are additionallypresent in several other organisms including Geobacter sulfurreducens(Coppi, Microbiology 151, 1239-1254 (2005)), Synechocystis str. PCC 6803(Germer, J. Biol. Chem., 284(52), 36462-36472 (2009)), and Thiocapsaroseopersicina (Rakhely, Appl. Environ. Microbiol. 70(2) 722-728(2004)). The Synechocystis enzyme is capable of generating NADPH fromhydrogen. Overexpression of both the Hox operon from Synechocystis str.PCC 6803 and the accessory genes encoded by the Hyp operon from Nostocsp. PCC 7120 led to increased hydrogenase activity compared toexpression of the Hox genes alone (Germer, J. Biol. Chem. 284(52),36462-36472 (2009)).

Protein GenBank ID GI Number Organism HoxF NP_942727.1 38637753Ralstonia eutropha H16 HoxU NP_942728.1 38637754 Ralstonia eutropha H16HoxY NP_942729.1 38637755 Ralstonia eutropha H16 HoxH NP_942730.138637756 Ralstonia eutropha H16 HoxW NP_942731.1 38637757 Ralstoniaeutropha H16 HoxI NP_942732.1 38637758 Ralstonia eutropha H16 HoxENP_953767.1 39997816 Geobacter sulfurreducens HoxF NP_953766.1 39997815Geobacter sulfurreducens HoxU NP_953765.1 39997814 Geobactersulfurreducens HoxY NP_953764.1 39997813 Geobacter sulfurreducens HoxHNP_953763.1 39997812 Geobacter sulfurreducens GSU2717 NP_953762.139997811 Geobacter sulfurreducens HoxE NP_441418.1 16330690Synechocystis str. PCC 6803 HoxF NP_441417.1 16330689 Synechocystis str.PCC 6803 Unknown NP_441416.1 16330688 Synechocystis str. PCC 6803function HoxU NP_441415.1 16330687 Synechocystis str. PCC 6803 HoxYNP_441414.1 16330686 Synechocystis str. PCC 6803 Unknown NP_441413.116330685 Synechocystis str. PCC 6803 function Unknown NP_441412.116330684 Synechocystis str. PCC 6803 function HoxH NP_441411.1 16330683Synechocystis str. PCC 6803 HypF NP_484737.1 17228189 Nostoc sp. PCC7120 HypC NP_484738.1 17228190 Nostoc sp. PCC 7120 HypD NP_484739.117228191 Nostoc sp. PCC 7120 Unknown NP_484740.1 17228192 Nostoc sp. PCC7120 function HypE NP_484741.1 17228193 Nostoc sp. PCC 7120 HypANP_484742.1 17228194 Nostoc sp. PCC 7120 HypB NP_484743.1 17228195Nostoc sp. PCC 7120 Hox1E AAP50519.1 37787351 Thiocapsa roseopersicinaHox1F AAP50520.1 37787352 Thiocapsa roseopersicina Hox1U AAP50521.137787353 Thiocapsa roseopersicina Hox1Y AAP50522.1 37787354 Thiocapsaroseopersicina Hox1H AAP50523.1 37787355 Thiocapsa roseopersicina

The genomes of E. coli and other enteric bacteria encode up to fourhydrogenase enzymes (Sawers, G., Antonie Van Leeuwenhoek 66:57-88(1994); Sawers et al., J Bacteria 164:1324-1331 (1985); Sawers andBoxer, Eur. J Biochem. 156:265-275 (1986); Sawers et al., J Bacteriol.168:398-404 (1986)). Given the multiplicity of enzyme activities E. colior another host organism can provide sufficient hydrogenase activity tosplit incoming molecular hydrogen and reduce the corresponding acceptor.Endogenous hydrogen-lyase enzymes of E. coli include hydrogenase 3, amembrane-bound enzyme complex using ferredoxin as an acceptor, andhydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4are encoded by the hyc and hyfgene clusters, respectively. Hydrogenaseactivity in E coli is also dependent upon the expression of the hypgenes whose corresponding proteins are involved in the assembly of thehydrogenase complexes (Jacobi et al., Arch. Microbiol 158:444-451(1992); Rangarajan et al., J Bacteriol 190:1447-1458 (2008)). The M.thermoacetica and Clostridium ljungdahli hydrogenases are suitable for ahost that lacks sufficient endogenous hydrogenase activity. M.thermoacetica and C. ljungdahli can grow with CO₂ as the exclusivecarbon source indicating that reducing equivalents are extracted from H₂to enable acetyl-CoA synthesis via the Wood-Ljungdahl pathway (Drake, H.L., J Bacteriol. 150:702-709 (1982); Drake and Daniel, Res Microbiol155:869-883 (2004); Kellum and Drake, J Bacteriol. 160:466-469 (1984)).M. thermoacetica has homologs to several hyp, hyc, and hyf genes from E.coli. These protein sequences encoded for by these genes are identifiedby the following GenBank accession numbers. In addition, several geneclusters encoding hydrogenase functionality are present in M.thermoacetica and C. ljungdahli (see for example US 2012/0003652).

Protein GenBank ID GI Number Organism HypA NP_417206 16130633Escherichia coli HypB NP_417207 16130634 Escherichia coli HypC NP_41720816130635 Escherichia coli HypD NP_417209 16130636 Escherichia coli HypENP_417210 226524740 Escherichia coli HypF NP_417192 16130619 Escherichiacoli HycA NP_417205 16130632 Escherichia coli HycB NP_417204 16130631Escherichia coli HycC NP_417203 16130630 Escherichia coli HycD NP_41720216130629 Escherichia coli HycE NP_417201 16130628 Escherichia coli HycFNP_417200 16130627 Escherichia coli HycG NP_417199 16130626 Escherichiacoli HycH NP_417198 16130625 Escherichia coli HycI NP_417197 16130624Escherichia coli HyfA NP_416976 90111444 Escherichia coli HyfB NP_41697716130407 Escherichia coli HyfC NP_416978 90111445 Escherichia coli HyfDNP_416979 16130409 Escherichia coli HyfE NP_416980 16130410 Escherichiacoli HyfF NP_416981 16130411 Escherichia coli HyfG NP_416982 16130412Escherichia coli HyfH NP_416983 16130413 Escherichia coli HyfI NP_41698416130414 Escherichia coli HyfJ NP_416985 90111446 Escherichia coli HyfRNP_416986 90111447 Escherichia coli

Proteins in M. thermoacetica whose genes are homologous to the E. colihydrogenase genes are shown below.

Protein GenBank ID GI Number Organism Moth_2175 YP_431007 83590998Moorella thermoacetica Moth_2176 YP_431008 83590999 Moorellathermoacetica Moth_2177 YP_431009 83591000 Moorella thermoaceticaMoth_2178 YP_431010 83591001 Moorella thermoacetica Moth_2179 YP_43101183591002 Moorella thermoacetica Moth_2180 YP_431012 83591003 Moorellathermoacetica Moth_2181 YP_431013 83591004 Moorella thermoaceticaMoth_2182 YP_431014 83591005 Moorella thermoacetica Moth_2183 YP_43101583591006 Moorella thermoacetica Moth_2184 YP_431016 83591007 Moorellathermoacetica Moth_2185 YP_431017 83591008 Moorella thermoaceticaMoth_2186 YP_431018 83591009 Moorella thermoacetica Moth_2187 YP_43101983591010 Moorella thermoacetica Moth_2188 YP_431020 83591011 Moorellathermoacetica Moth_2189 YP_431021 83591012 Moorella thermoaceticaMoth_2190 YP_431022 83591013 Moorella thermoacetica Moth_2191 YP_43102383591014 Moorella thermoacetica Moth_2192 YP_431024 83591015 Moorellathermoacetica Moth_0439 YP_429313 83589304 Moorella thermoaceticaMoth_0440 YP_429314 83589305 Moorella thermoacetica Moth_0441 YP_42931583589306 Moorella thermoacetica Moth_0442 YP_429316 83589307 Moorellathermoacetica Moth_0809 YP_429670 83589661 Moorella thermoaceticaMoth_0810 YP_429671 83589662 Moorella thermoacetica Moth_0811 YP_42967283589663 Moorella thermoacetica Moth_0812 YP_429673 83589664 Moorellathermoacetica Moth_0814 YP_429674 83589665 Moorella thermoaceticaMoth_0815 YP_429675 83589666 Moorella thermoacetica Moth_0816 YP_42967683589667 Moorella thermoacetica Moth_1193 YP_430050 83590041 Moorellathermoacetica Moth_1194 YP_430051 83590042 Moorella thermoaceticaMoth_1195 YP_430052 83590043 Moorella thermoacetica Moth_1196 YP_43005383590044 Moorella thermoacetica Moth_1717 YP_430562 83590553 Moorellathermoacetica Moth_1718 YP_430563 83590554 Moorella thermoaceticaMoth_1719 YP_430564 83590555 Moorella thermoacetica Moth_1883 YP_43072683590717 Moorella thermoacetica Moth_1884 YP_430727 83590718 Moorellathermoacetica Moth_1885 YP_430728 83590719 Moorella thermoaceticaMoth_1886 YP_430729 83590720 Moorella thermoacetica Moth_1887 YP_43073083590721 Moorella thermoacetica Moth_1888 YP_430731 83590722 Moorellathermoacetica Moth_1452 YP_430305 83590296 Moorella thermoaceticaMoth_1453 YP_430306 83590297 Moorella thermoacetica Moth_1454 YP_43030783590298 Moorella thermoacetica

Genes encoding hydrogenase enzymes from C. ljungdahli are shown below.

Protein GenBank ID GI Number Organism CLJU_c20290 ADK15091.1 300435324Clostridium ljungdahli CLJU_c07030 ADK13773.1 300434006 Clostridiumljungdahli CLJU_c07040 ADK13774.1 300434007 Clostridium ljungdahliCLJU_c07050 ADK13775.1 300434008 Clostridium ljungdahli CLJU_c07060ADK13776.1 300434009 Clostridium ljungdahli CLJU_c07070 ADK13777.1300434010 Clostridium ljungdahli CLJU_c07080 ADK13778.1 300434011Clostridium ljungdahli CLJU_c14730 ADK14541.1 300434774 Clostridiumljungdahli CLJU_c14720 ADK14540.1 300434773 Clostridium ljungdahliCLJU_c14710 ADK14539.1 300434772 Clostridium ljungdahli CLJU_c14700ADK14538.1 300434771 Clostridium ljungdahli CLJU_c28670 ADK15915.1300436148 Clostridium ljungdahli CLJU_c28660 ADK15914.1 300436147Clostridium ljungdahli CLJU_c28650 ADK15913.1 300436146 Clostridiumljungdahli CLJU_c28640 ADK15912.1 300436145 Clostridium ljungdahli

In some cases, hydrogenase encoding genes are located adjacent to aCODH. In Rhodospirillum rubrum, the encoded CODH/hydrogenase proteinsform a membrane-bound enzyme complex that has been indicated to be asite where energy, in the form of a proton gradient, is generated fromthe conversion of CO and H₂O to CO₂ and H2 (Fox et al., J Bacteriol178:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and itsadjacent genes have been proposed to catalyze a similar functional rolebased on their similarity to the R rubrum CODH/hydrogenase gene cluster(Wu et al., PLoS Genet. 1:e65 (2005)). The C. hydrogenoformans CODH-Iwas also shown to exhibit intense CO oxidation and CO₂ reductionactivities when linked to an electrode (Parkin et al., J Am. Chem. Soc.129:10328-10329 (2007)).

Protein GenBank ID GI Number Organism CooL AAC45118 1515468Rhodospirillum rubrum CooX AAC45119 1515469 Rhodospirillum rubrum CooUAAC45120 1515470 Rhodospirillum rubrum CooH AAC45121 1498746Rhodospirillum rubrum CooF AAC45122 1498747 Rhodospirillum rubrum CODH(CooS) AAC45123 1498748 Rhodospirillum rubrum CooC AAC45124 1498749Rhodospirillum rubrum CooT AAC45125 1498750 Rhodospirillum rubrum CooJAAC45126 1498751 Rhodospirillum rubrum CODH-I (CooS-I) YP_36064478043418 Carboxydothermus hydrogenoformans CooF YP_360645 78044791Carboxydothermus hydrogenoformans HypA YP_360646 78044340Carboxydothermus hydrogenoformans CooH YP_360647 78043871Carboxydothermus hydrogenoformans CooU YP_360648 78044023Carboxydothermus hydrogenoformans CooX YP_360649 78043124Carboxydothermus hydrogenoformans CooL YP_360650 78043938Carboxydothermus hydrogenoformans CooK YP_360651 78044700Carboxydothermus hydrogenoformans CooM YP_360652 78043942Carboxydothermus hydrogenoformans CooC YP_360654.1 78043296Carboxydothermus hydrogenoformans CooA-1 YP_360655.1 78044021Carboxydothermus hydrogenoformans

Some hydrogenase and CODH enzymes transfer electrons to ferredoxinsFerredoxins are small acidic proteins containing one or more iron-sulfurclusters that function as intracellular electron Gathers with a lowreduction potential. Reduced ferredoxins donate electrons toFe-dependent enzymes such as ferredoxin-NADP⁺ oxidoreductase,pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxinoxidoreductase (OFOR). The H. thermophilus gene fdx1 encodes a[4Fe-4S]-type ferredoxin that is required for the reversiblecarboxylation of 2-oxoglutarate and pyruvate by OFOR and PFOR,respectively (Yamamoto et al., Extremophiles 14:79-85 (2010)). Theferredoxin associated with the Sulfolobus solfataricus2-oxoacid:ferredoxin reductase is a monomeric dicluster[3Fe-4S][4Fe-4S]type ferredoxin (Park et al. 2006). While the geneassociated with this protein has not been fully sequenced, theN-terminal domain shares 93% homology with the zfx ferredoxin from S.acidocaldarius. The E. coli genome encodes a soluble ferredoxin ofunknown physiological function, fdx. Some evidence indicates that thisprotein can function in iron-sulfur cluster assembly (Takahashi andNakamura, 1999). Additional ferredoxin proteins have been characterizedin Helicobacter pylori (Mukhopadhyay et al. 2003) and Campylobacterjejuni (van Vliet et al. 2001). A 2Fe-2S ferredoxin from Clostridiumpasteurianum has been cloned and expressed in E. coli (Fujinaga andMeyer, Biochemical and Biophysical Research Communications, 192(3):(1993)). Acetogenic bacteria such as Moorella thermoacetica, Clostridiumcarboxidivorans P7, Clostridium ljungdahli and Rhodospirillum rubrum arepredicted to encode several ferredoxins, listed below.

Protein GenBank ID GI Number Organism fdx1 BAE02673.1 68163284Hydrogenobacter thermophilus M11214.1 AAA83524.1 144806 Clostridiumpasteurianum Zfx AAY79867.1 68566938 Sulfolobus acidocalarius FdxAAC75578.1 1788874 Escherichia coli hp 0277 AAD07340.1 2313367Helicobacter pylori fdxA CAL34484.1 112359698 Campylobacter jejuniMoth_0061 ABC18400.1 83571848 Moorella thermoacetica Moth_1200ABC19514.1 83572962 Moorella thermoacetica Moth_1888 ABC20188.1 83573636Moorella thermoacetica Moth_2112 ABC20404.1 83573852 Moorellathermoacetica Moth_1037 ABC19351.1 83572799 Moorella thermoaceticaCcarbDRAFT_4383 ZP_05394383.1 255527515 Clostridium carboxidivorans P7CcarbDRAFT_2958 ZP_05392958.1 255526034 Clostridium carboxidivorans P7CcarbDRAFT_2281 ZP_05392281.1 255525342 Clostridium carboxidivorans P7CcarbDRAFT_5296 ZP_05395295.1 255528511 Clostridium carboxidivorans P7CcarbDRAFT_1615 ZP_05391615.1 255524662 Clostridium carboxidivorans P7CcarbDRAFT_1304 ZP_05391304.1 255524347 Clostridium carboxidivorans P7cooF AAG29808.1 11095245 Carboxydothermus hydrogenoformans fdxNCAA35699.1 46143 Rhodobacter capsulatus Rru_A2264 ABC23064.1 83576513Rhodospirillum rubrum Rru_A1916 ABC22716.1 83576165 Rhodospirillumrubrum Rru_A2026 ABC22826.1 83576275 Rhodospirillum rubrum cooFAAC45122.1 1498747 Rhodospirillum rubrum fdxN AAA26460.1 152605Rhodospirillum rubrum Alvin_2884 ADC63789.1 288897953 Allochromatiumvinosum DSM 180 Fdx YP_002801146.1 226946073 Azotobacter vinelandii DJCKL_3790 YP_001397146.1 153956381 Clostridium kluyveri DSM 555 fer1NP_949965.1 39937689 Rhodopseudomonas palustris CGA009 Fdx CAA12251.13724172 Thauera aromatica CHY_2405 YP_361202.1 78044690 Carboxydothermushydrogenoformans Fer YP_359966.1 78045103 Carboxydothermushydrogenoformans Fer AAC83945.1 1146198 Bacillus subtilis fdx1NP_249053.1 15595559 Pseudomonas aeruginosa PA01 yfhL AP 003148.189109368 Escherichia coli K-12 CLJU_c00930 ADK13195.1 300433428Clostridium ljungdahli CLJU_c00010 ADK13115.1 300433348 Clostridiumljungdahli CLJU_c01820 ADK13272.1 300433505 Clostridium ljungdahliCLJU_c17980 ADK14861.1 300435094 Clostridium ljungdahli CLJU_c17970ADK14860.1 300435093 Clostridium ljungdahli CLJU_c22510 ADK15311.1300435544 Clostridium ljungdahli CLJU_c26680 ADK15726.1 300435959Clostridium ljungdahli CLJU_c29400 ADK15988.1 300436221 Clostridiumljungdahli

Ferredoxin oxidoreductase enzymes transfer electrons from ferredoxins orflavodoxins to NAD(P)H. Two enzymes catalyzing the reversible transferof electrons from reduced ferredoxins to NAD(P)+ are ferredoxin:NAD+oxidoreductase (EC 1.18.1.3) and ferredoxin:NADP+ oxidoreductase (FNR,EC 1.18.1.2). Ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) has anoncovalently bound FAD cofactor that facilitates the reversibletransfer of electrons from NADPH to low-potential acceptors such asferredoxins or flavodoxins.

Protein GenBank ID GI Number Organism fqrB NP_207955.1 15645778Helicobacter pylori fqrB YP_001482096.1 157414840 Campylobacter jejuniRPA3954 CAE29395.1 39650872 Rhodopseudomonas palustris Fpr BAH29712.1225320633 Hydrogenobacter thermophilus yumC NP_3910912 255767736Bacillus subtilis Fpr P28861.4 399486 Escherichia coli hcaD AAC75595.11788892 Escherichia coli LOC100282643 NP_001149023.1 226497434 Zea maysNfnA YP_001393861.1 153953096 Clostridium kluyveri NfnB YP_001393862.1153953097 Clostridium kluyveri CcarbDRAFT_2639 ZP_05392639.1 255525707Clostridium carboxidivorans P7 CcarbDRAFT_2638 ZP_05392638.1 255525706Clostridium carboxidivorans P7 CcarbDRAFT_2636 ZP_05392636.1 255525704Clostridium carboxidivorans P7 CcarbDRAFT_5060 ZP_05395060.1 255528241Clostridium carboxidivorans P7 CcarbDRAFT_2450 ZP_05392450.1 255525514Clostridium carboxidivorans P7 CcarbDRAFT_1084 ZP_05391084.1 255524124Clostridium carboxidivorans P7 RnfC EDK33306.1 146346770 Clostridiumkluyveri RnfD EDK33307.1 146346771 Clostridium kluyveri RnfG EDK33308.1146346772 Clostridium kluyveri RnfE EDK33309.1 146346773 Clostridiumkluyveri RnfA EDK33310.1 146346774 Clostridium kluyveri RnfB EDK33311.1146346775 Clostridium kluyveri CLJU_c11410 (RnfB) ADK14209.1 300434442Clostridium ljungdahlii CLJU_c11400 (RnfA) ADK14208.1 300434441Clostridium ljungdahlii CLJU_c11390 (RnfE) ADK14207.1 300434440Clostridium ljungdahlii CLJU_c11380 (RnfG) ADK14206.1 300434439Clostridium ljungdahlii CLJU_c11370 (RnfD) ADK14205.1 300434438Clostridium ljungdahlii CLJU_c11360 (RnfC) ADK14204.1 300434437Clostridium ljungdahlii MOTH_1518 (NfhA) YP_430370.1 83590361 Moorellathermoacetica MOTH_1517(NfhB) YP_430369.1 83590360 Moorellathermoacetica CHY_1992 (NfhA) YP_360811.1 78045020 Carboxydothermushydrogenoformans CHY_1993 (NfhB) YP_360812.1 78044266 Carboxydothermushydrogenoformans CLJU_c37220 (NfnAB) YP_003781850.1 300856866Clostridium ljungdahlii

FIG. 2, Step I—Formate Dehydrogenase

Formate dehydrogenase (FDH) catalyzes the reversible transfer ofelectrons from formate to an acceptor. Exemplary enzymes include thosedescribed in the section for Step S, FIG. 1: Formate dehydrogenase.

FIG. 2, Step J—Methanol Dehydrogenase

NAD+ dependent methanol dehydrogenase enzymes (EC 1.1.1.244) catalyzethe conversion of methanol and NAD+ to formaldehyde and NADH. An enzymewith this activity was first characterized in Bacillus methanolicus(Heggeset, et al., Applied and Environmental Microbiology,78(15):5170-5181 (2012)). This enzyme is zinc and magnesium dependent,and activity of the enzyme is enhanced by the activating enzyme encodedby act (Kloosterman et al, J Biol Chem 277:34785-92 (2002)). The act isa Nudix hydrolase. Several of these candidates have been identified andshown to have activity on methanol. Additional NAD(P)+ dependent enzymescan be identified by sequence homology. Methanol dehydrogenase enzymesutilizing different electron acceptors are also known in the art.Examples include cytochrome dependent enzymes such as mxalF of themethylotroph Methylobacterium extorquens (Nunn et al, Nucl Acid Res16:7722 (1988)). Methanol dehydrogenase enzymes of methanotrophs such asMethylococcus capsulatis function in a complex with methanemonooxygenase (MMO) (Myronova et al, Biochem 45:11905-14 (2006)).Methanol can also be oxidized to formaldehyde by alcohol oxidase enzymessuch as methanol oxidase (EC 1.1.3.13) of Candida boidinii (Sakai et al,Gene 114: 67-73 (1992)).

Protein GenBank ID GI Number Organism mdh, MGA3_17392 EIJ77596.1387585261 Bacillus methanolicus MGA3 mdh2, MGA3_07340 EIJ83020.1387590701 Bacillus methanolicus MGA3 mdh3, MGA3_10725 EIJ80770.1387588449 Bacillus methanolicus MGA3 act, MGA3_09170 EIJ83380.1387591061 Bacillus methanolicus MGA3 mdh, PB1_17533 ZP_10132907.1387930234 Bacillus methanolicus PB1 mdh1, PB1_14569 ZP_10132325.1387929648 Bacillus methanolicus PB1 mdh2, PB1_12584 ZP_10131932.1387929255 Bacillus methanolicus PB1 act, PB1_14394 ZP_10132290.1387929613 Bacillus methanolicus PB1 BFZC1_05383 ZP_07048751.1 299535429Lysinibacillus fusiformis BFZC1_20163 ZP_07051637.1 299538354Lysinibacillus fusiformis Bsph_4187 YP_001699778.1 169829620Lysinibacillus sphaericus Bsph_1706 YP_001697432.1 169827274Lysinibacillus sphaericus mdh2 YP_004681552.1 339322658 Cupriavidusnecator N-1 nudF1 YP_004684845.1 339325152 Cupriavidus necator N-1BthaA_010200007655 ZP_05587334.1 257139072 Burkholderia thailandensisE264 BTH_I1076 YP_441629.1 83721454 Burkholderia thailandensis E264(MutT/NUDIX NTP pyrophosphatase) BalcAV_11743 ZP_10819291.1 402299711Bacillus alcalophilus ATCC 27647 BalcAV_05251 ZP_10818002.1 402298299Bacillus alcalophilus ATCC 27647 alcohol dehydrogenase YP_001447544156976638 Vibrio harveyi ATCC BAA-1116 P3TCK_27679 ZP_01220157.190412151 Photobacterium profundum 3TCK alcohol dehydrogenase YP_694908110799824 Clostridium perfringens ATCC 13124 adhB NP_717107 24373064Shewanella oneidensis MR-1 alcohol dehydrogenase YP_237055 66047214Pseudomonas syringae pv. syringae B728a alcohol dehydrogenase YP_35977278043360 Carboxydothermus hydrogenoformans Z-2901 alcohol dehydrogenaseYP_003990729 312112413 Geobacillus sp. Y4.1MC1 PpeoK3_010100018471ZP_10241531.1 390456003 Paenibacillus peoriae KCTC 3763 OBE_12016EKC54576 406526935 human gut metagenome alcohol dehydrogenaseYP_001343716 152978087 Actinobacillus succinogenes 130Z dhaT AAC456512393887 Clostridium pasteurianum DSM 525 alcohol dehydrogenase NP_56185218309918 Clostridium perfringens str. 13 BAZO_10081 ZP_11313277.1410459529 Bacillus azotoformans LMG 9581 alcohol dehydrogenaseYP_007491369 452211255 Methanosarcina mazei Tuc01 alcohol dehydrogenaseYP_004860127 347752562 Bacillus coagulans 36D1 alcohol dehydrogenaseYP_002138168 197117741 Geobacter bemidjiensis Bem DesmeDRAFT_1354ZP_08977641.1 354558386 Desulfitobacterium metallireducens DSM 15288alcohol dehydrogenase YP_001337153 152972007 Klebsiella pneumoniaesubsp. pneumoniae MGH 78578 alcohol dehydrogenase YP_001113612 134300116Desulfotomaculum reducens MI-1 alcohol dehydrogenase YP_001663549167040564 Thermoanaerobacter sp. X514 ACINNAV82_2382 ZP_16224338.1421788018 Acinetobacter baumannii Naval-82 alcohol dehydrogenaseYP_005052855 374301216 Desulfovibrio africanus str. Walvis Bay alcoholdehydrogenase AGF87161 451936849 uncultured organism DesfrDRAFT_3929ZP_07335453.1 303249216 Desulfovibrio fructosovorans JJ alcoholdehydrogenase NP_617528 20091453 Methanosarcina acetivorans C2A alcoholdehydrogenase NP_343875.1 15899270 Sulfolobus solfataricus P-2 adh4YP_006863258 408405275 Nitrososphaera gargensis Ga9.2 Ta0841 NP_394301.116081897 Thermoplasma acidophilum PTO1151 YP_023929.1 48478223Picrophilus torridus DSM9790 alcohol dehydrogenase ZP_10129817.1387927138 Bacillus methanolicus PB-1 cgR_2695 YP_001139613.1 145296792Corynebacterium glutamicum R alcohol dehydrogenase YP_004758576.1340793113 Corynebacterium variabile HMPREF1015_01790 ZP_09352758.1365156443 Bacillus smithii ADH1 NP_014555.1 6324486 Saccharomycescerevisiae NADH-dependent YP_001126968.1 138896515 Geobacillusthemodenitrificans NG80-2 butanol dehydrogenase A alcohol dehydrogenaseWP_007139094.1 494231392 Flavobacterium frigoris methanol dehydrogenaseWP_003897664.1 489994607 Mycobacterium smegmatis ADH1B NP_000659.234577061 Homo sapiens PMI01_01199 ZP_10750164.1 399072070 Caulobactersp. AP07 YiaY YP_026233.1 49176377 Escherichia coli MCA0299 YP_112833.153802410 Methylococcus capsulatis MCA0782 YP_113284.1 53804880Methylococcus capsulatis mxaI YP_002965443.1 240140963 Methylobacteriumextorquens mxaF YP_002965446.1 240140966 Methylobacterium extorquensAOD1 AAA34321.1 170820 Candida boidinii hypothetical protein EDA87976.1142827286 Marine metagenome JCVI_SCAF_1096627185304 GOS_1920437 alcoholdehydrogenase CAA80989.1 580823 Geobacillus stearothermophilus

An in vivo assay was developed to determine the activity of methanoldehydrogenases. This assay relies on the detection of formaldehyde(HCHO), thus measuring the forward activity of the enzyme (oxidation ofmethanol). To this end, a strain comprising a BDOP and lacking frmA,frmB, frmR was created using Lambda Red recombinase technology (Datsenkoand Wanner, Proc. Natl. Acad. Sci. USA, 6 97(12): 6640-5 (2000).Plasmids expressing methanol dehydrogenases were transformed into thestrain, then grown to saturation in LB medium+antibiotic at 37° C. withshaking. Transformation of the strain with an empty vector served as anegative control. Cultures were adjusted by O.D. and then diluted 1:10into M9 medium+0.5% glucose+antibiotic and cultured at 37° C. withshaking for 6-8 hours until late log phase. Methanol was added to 2% v/vand the cultures were further incubated for 30 min. with shaking at 37°C. Cultures were spun down and the supernatant was assayed forformaldehyde produced using DETECTX Formaldehyde Detection kit (ArborAssays; Ann Arbor, Mich.) according to manufacturer's instructions. ThefrmA, frmB, frmR deletions resulted in the native formaldehydeutilization pathway to be deleted, which enables the formation offormaldehyde that can be used to detect methanol dehydrogenase activityin the non-naturally occurring microbial organism.

The activity of several enzymes was measured using the assay describedabove. The results of four independent experiments are provided in thebelow table.

Results of in vivo assays showing formaldehyde (HCHO) production byvarious non-naturally occurring microbial organism comprising a plasmidexpressing a methanol dehydrogenase.

HCHO Accession number (μM) Experiment 1 EIJ77596.1 >50 EIJ83020.1 >20EIJ80770.1 >50 ZP_10132907.1 >20 ZP_10132325.1 >20 ZP_10131932.1 >50ZP_07048751.1 >50 YP_001699778.1 >50 YP_004681552.1 >10 ZP_10819291.1 <1Empty vector 2.33 Experiment 2 EIJ77596.1 >50 NP_00659.2 >50YP_004758576.1 >20 ZP_09352758.1 >50 ZP_10129817.1 >20YP_001139613.1 >20 NP_014555.1 >10 WP_007139094.1 >10 NP_343875.1 >1YP_006863258 >1 NP_394301.1 >1 ZP_10750164.1 >1 YP_023929.1 >1ZP_08977641.1 <1 ZP_10117398.1 <1 YP_004108045.1 <1 ZP_09753449.1 <1Empty vector 0.17 Experiment 3 EIJ77596.1 >50 NP_561852 >50YP_002138168 >50 YP_026233.1 >50 YP_001447544 >50 Metalibrary >50YP_359772 >50 ZP_01220157.1 >50 ZP_07335453.1 >20 YP_001337153 >20YP_694908 >20 NP_717107 >20 AAC45651 >10 ZP_11313277.1 >10ZP_16224338.1 >10 YP_001113612 >10 YP_004860127 >10 YP_003310546 >10YP_001343716 >10 NP_717107 >10 YP_002434746 >10 Empty vector 0.11Experiment 4 EIJ77596.1 >20 ZP_11313277.1 >50 YP_001113612 >50YP_001447544 >20 AGF87161 >50 EDA87976.1 >20 Empty vector −0.8

FIG. 2, Step K—Spontaneous or Formaldehyde Activating Enzyme

The conversion of formaldehyde and THF to methylenetetrahydrofolate canoccur spontaneously. It is also possible that the rate of this reactioncan be enhanced by a formaldehyde activating enzyme. A formaldehydeactivating enzyme (Fae) has been identified in Methylobacteriumextorquens AM1 which catalyzes the condensation of formaldehyde andtetrahydromethanopterin to methylene tetrahydromethanopterin (Vorholt,et al., J. Bacteriol., 182(23), 6645-6650 (2000)). It is possible that asimilar enzyme exists or can be engineered to catalyze the condensationof formaldehyde and tetrahydrofolate to methylenetetrahydrofolate.Homologs exist in several organisms.

Protein GenBank ID GI Number Organism MexAM1_META1p1766 Q9FA38.317366061 Methylobacterium extorquens AM1 Xaut_0032 YP_001414948.1154243990 Xanthobacter autotrophicus Py2 Hden_1474 YP_003755607.1300022996 Hyphomicrobium denitrificans ATCC 51888

FIG. 2, Step L—Formaldehyde Dehydrogenase

Oxidation of formaldehyde to formate is catalyzed by formaldehydedehydrogenase. An NAD+ dependent formaldehyde dehydrogenase enzyme isencoded by fdhA of Pseudomonas putida (Ito et al, J Bacteriol 176:2483-2491 (1994)). Additional formaldehyde dehydrogenase enzymes includethe NAD+ and glutathione independent formaldehyde dehydrogenase fromHyphomicrobium zavarzinii (Jerome et al, Appl Microbiol Biotechnol77:779-88 (2007)), the glutathione dependent formaldehyde dehydrogenaseof Pichia pastoris (Sunga et al, Gene 330:39-47 (2004)) and the NAD(P)+dependent formaldehyde dehydrogenase of Methylobacter marinus (Speer etal, FEMS Microbiol Lett, 121(3):349-55 (1994)).

Protein GenBank ID GI Number Organism fdhA P46154.3 1169603 Pseudomonasputida faoA CAC85637.1 19912992 Hyphomicrobium zavarzinii Fld1CCA39112.1 328352714 Pichia pastoris fdh P47734.2 221222447Methylobacter marinus

In addition to the formaldehyde dehydrogenase enzymes listed above,alternate enzymes and pathways for converting formaldehyde to formateare known in the art. For example, many organisms employglutathione-dependent formaldehyde oxidation pathways, in whichformaldehyde is converted to formate in three steps via theintermediates S-hydroxymethylglutathione and S-formylglutathione(Vorholt et al, J Bacteriol 182:6645-50 (2000)). The enzymes of thispathway are S-(hydroxymethyl)glutathione synthase (EC 4.4.1.22),glutathione-dependent formaldehyde dehydrogenase (EC 1.1.1.284) andS-formylglutathione hydrolase (EC 3.1.2.12).

FIG. 2, Step M—Spontaneous or S-(Hydroxymethyl)Glutathione Synthase

While conversion of formaldehyde to S-hydroxymethylglutathione can occurspontaneously in the presence of glutathione, it has been shown byGoenrich et al (Goenrich, et al., J Biol Chem 277(5); 3069-72 (2002))that an enzyme from Paracoccus denitrificans can accelerate thisspontaneous condensation reaction. The enzyme catalyzing the conversionof formaldehyde and glutathione was purified and namedglutathione-dependent formaldehyde-activating enzyme (Gfa). Putativeproteins with sequence identity to Gfa from P. denitrificans are presentalso in Rhodobacter sphaeroides, Sinorhizobium meliloti, andMesorhizobium loti.

Protein GenBank ID GI Number Organism Gfa Q51669.3 38257308 Paracoccusdenitrificans Gfa ABP71667.1 145557054 Rhodobacter sphaeroides ATCC17025 Gfa Q92WX6.1 38257348 Sinorhizobium meliloti 1021 Gfa Q98LU4.238257349 Mesorhizobium loti MAFF303099

FIG. 2, Step N—Glutathione-Dependent Formaldehyde Dehydrogenase

Glutathione-dependent formaldehyde dehydrogenase (GS-FDH) belongs to thefamily of class DI alcohol dehydrogenases. Glutathione and formaldehydecombine non-enzymatically to form hydroxymethylglutathione, the truesubstrate of the GS-FDH catalyzed reaction. The product,S-formylglutathione, is further metabolized to formic acid.

Protein GenBank ID GI Number Organism frmA YP_488650.1 388476464Escherichia coli K-12 MG1655 SFA1 NP_010113.1 6320033 Saccharomycescerevisiae S288c flhA AAC44551.1 1002865 Paracoccus denitrificans adhIAAB09774.1 986949 Rhodobacter sphaeroidesFIG. 2, Step O—S-Formylglutathione Hydrolase S-formylglutathionehydrolase is a glutathione thiol esterase found in bacteria, plants andanimals. It catalyzes conversion of S-formylglutathione to formate andglutathione. Exemplary enzymes are below. YeiG of E. coli is apromiscuous serine hydrolase; its highest specific activity is with thesubstrate S-formylglutathione.

Protein GenBank ID GI Number Organism frmB NP_414889.1 16128340Escherichia coli K-12 MG1655 yeiG AAC75215.1 1788477 Escherichia coliK-12 MG1655 fghA AAC44554.1 1002868 Paracoccus denitrificans

FIG. 2, Step P—Carbon Monoxide Dehydrogenase (CODH)

CODH is a reversible enzyme that interconverts CO and CO₂ at the expenseor gain of electrons. The natural physiological role of the CODH inACS/CODH complexes is to convert CO₂ to CO for incorporation intoacetyl-CoA by acetyl-CoA synthase. Nevertheless, such CODH enzymes aresuitable for the extraction of reducing equivalents from CO due to thereversible nature of such enzymes. Expressing such CODH enzymes in theabsence of ACS allows them to operate in the direction opposite to theirnatural physiological role (i.e., CO oxidation).

In M. thermoacetica, C. hydrogenoformans, C. carboxidivorans P7, andseveral other organisms, additional CODH encoding genes are locatedoutside of the ACS/CODH operons. These enzymes provide a means forextracting electrons (or reducing equivalents) from the conversion ofcarbon monoxide to carbon dioxide. The M. thermoacetica gene (GenbankAccession Number YP_430813) is expressed by itself in an operon and isbelieved to transfer electrons from CO to an external mediator likeferredoxin in a “Ping-pong” reaction. The reduced mediator then couplesto other reduced nicolinamide adenine dinucleotide phosphate (NAD(P)H)carriers or ferredoxin-dependent cellular processes (Ragsdale, Annals ofthe New York Academy of Sciences 1125: 129-136 (2008)). Similar ACS-freeCODH enzymes can be found in a diverse army of organisms.

Protein GenBank ID GI Number Organism CODH (putative) YP_430813 83590804Moorella thermoacetica CODH-II (CooS-II) YP_358957 78044574Carboxydothermus hydrogenoformans CooF YP_358958 78045112Carboxydothermus hydrogenoformans CODH (putative) ZP_05390164.1255523193 Clostridium carboxidivorans P7 CcarbDRAFT_0341 ZP_05390341.1255523371 Clostridium carboxidivorans P7 CcarbDRAFT_1756 ZP_05391756.1255524806 Clostridium carboxidivorans P7 CcarbDRAFT_2944 ZP_05392944.1255526020 Clostridium carboxidivorans P7 CODH YP_384856.1 78223109Geobacter metallireducens GS-15 Cpha266_0148 (cytochrome c) YP_910642.1119355998 Chlorobium phaeobacteroides DSM 266 Cpha266_0149 (CODH)YP_910643.1 119355999 Chlorobium phaeobacteroides DSM 266 Ccel_0438YP_002504800.1 220927891 Clostridium cellulolyticum H10 Ddes_0382 (CODH)YP_002478973.1 220903661 Desulfovibrio desulfuricans subsp.desulfuricans str. ATCC 27774 Ddes_0381 (CooC) YP_002478972.1 220903660Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774Pcar_0057 (CODH) YP_355490.1 7791767 Pelobacter carbinolicus DSM 2380Pcar 0058 (CooC) YP_355491.1 7791766 Pelobacter carbinolicus DSM 2380Pcar_0058 (HypA) YP_355492.1 7791765 Pelobacter carbinolicus DSM 2380CooS (CODH) YP_001407343.1 154175407 Campylobacter curvus 525.92CLJU_c09110 ADK13979.1 300434212 Clostridium ljungdahli CLJU_c09100ADK13978.1 300434211 Clostridium ljungdahli CLJU c09090 ADK13977.1300434210 Clostridium ljungdahli

Example III Methods for Formaldehyde Fixation

Provided herein are exemplary pathways, which utilize formaldehydeproduced from the oxidation of methanol (see, e.g., FIG. 1, step A, orFIG. 2, step J) or from formate assimilation pathways described inExample I (see, e.g., FIG. 1) in the formation of intermediates ofcertain central metabolic pathways that can be used for the productionof compounds disclosed herein.

One exemplary pathway that can utilize formaldehyde produced from theoxidation of methanol is shown in FIG. 1, which involves condensation offormaldehyde and D-ribulose-5-phosphate to form hexulose-6-phosphate(h6p) by hexulose-6-phosphate synthase (FIG. 1, step B). The enzyme canuse Mg′ or Me for maximal activity, although other metal ions areuseful, and even non-metal-ion-dependent mechanisms are contemplated.H6p is converted into fructose-6-phosphate by6-phospho-3-hexuloisomerase (FIG. 1, step C).

Another exemplary pathway that involves the detoxification andassimilation of formaldehyde produced from the oxidation of methanol isshown in FIG. 1 and proceeds through dihydroxyacetone. Dihydroxyacetonesynthase is a special transketolase that first transfers a glycoaldehydegroup from xylulose-5-phosphate to formaldehyde, resulting in theformation of dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate(G3P), which is an intermediate in glycolysis (FIG. 1). The DHA obtainedfrom DHA synthase can be further phosphorylated to form DHA phosphateand assimilated into glycolysis and several other pathways (FIG. 1).Alternatively, or in addition, a fructose-6-phosphate aldolase can beused to catalyze the conversion of DHA and G3P to fructose-6-phosphate(FIG. 1, step Z).

FIG. 1, Steps B and C—Hexulose-6-Phosphate Synthase (Step B) and6-Phospho-3-Hexuloisomerase (Step C)

Both of the hexulose-6-phosphate synthase and6-phospho-3-hexuloisomerase enzymes are found in several organisms,including methanotrophs and methylotrophs where they have been. Inaddition, these enzymes have been reported in heterotrophs such asBacillus subtilis also where they are reported to be involved informaldehyde detoxification. Genes for these two enzymes from themethylotrophic bacterium Mycobacterium gastri MB19 have been fused andE. coli strains harboring the hps-phi construct showed more efficientutilization of formaldehyde (Orita et at, 2007, Appl MicrobiolBiotechnol. 76:439-445). In some organisms, these two enzymes naturallyexist as a fused version that is bifunctional.

Exemplary candidate genes for hexulose-6-phopshate synthase are:

Protein GenBank ID GI number Organism Hps AAR39392.1 40074227 Bacillusmethanolicus MGA3 Hps EIJ81375.1 387589055 Bacillus methanolicus PB1RmpA BAA83096.1 5706381 Methylomonas aminofaciens RmpA BAA90546.16899861 Mycobacterium gastri YckG BAA08980.1 1805418 Bacillus subtilisHps YP_544362.1 91774606 Methylobacillus flagellatus Hps YP_545763.191776007 Methylobacillus flagellatus Hps AAG29505.1 11093955 Aminomonasaminovorus SgbH YP_004038706.1 313200048 Methylovorus sp. MP688 HpsYP_003050044.1 253997981 Methylovorus glucosetrophus SIP3-4 HpsYP_003990382.1 312112066 Geobacillus sp. Y4.1MC1 Hps gb|AAR91478.140795504 Geobacillus sp. M10EXG Hps YP_007402409.1 448238351 Geobacillussp. GHH01

Exemplary gene candidates for 6-phospho-3-hexuloisomerase are:

Protein GenBank ID GI number Organism Phi AAR39393.1 40074228 Bacillusmethanolicus MGA3 Phi EIJ81376.1 387589056 Bacillus methanolicus PB1 PhiBAA83098.1 5706383 Methylomonas aminofaciens RmpB BAA90545.1 6899860Mycobacterium gastri Phi YP_545762.1 91776006 Methylobacillusflagellatus KT Phi YP_003051269.1 253999206 Methylovorus glucosetrophusSIP3-4 Phi YP_003990383.1 312112067 Geobacillus sp. Y4.1MC1 PhiYP_007402408.1 448238350 Geobacillus sp. GHH01

Candidates for enzymes where both of these functions have been fusedinto a single open reading frame include the following.

Protein GenBank ID GI number Organism PH1938 NP_143767.1 14591680Pyrococcus horikoshii OT3 PF0220 NP_577949.1 18976592 Pyrococcusfuriosus TK0475 YP_182888.1 57640410 Thermococcus kodakaraensis PAB1222NP_127388.1 14521911 Pyrococcus abyssi MCA2738 YP_115138.1 53803128Methylococcus capsulatas Metal_3152 EIC30826.1 380884949Methylomicrobium album BG8

FIG. 1, Step D—Dihydroxyacetone Synthase

The dihydroxyacetone synthase enzyme in Candida boidinii uses thiaminepyrophosphate and Mg²⁺ as cofactors and is localized in the peroxisome.The enzyme from the methanol-growing carboxydobacterium, Mycobacter sp.strain JC1 DSM 3803, was also found to have DHA synthase and kinaseactivities. Several other mycobacteria, excluding only Mycobacteriumtuberculosis, can use methanol as the sole source of carbon and energyand are reported to use dihydroxyacetone synthase.

Protein GenBank ID GI number Organism DAS1 AAC83349.1 3978466 Candidaboidinii HPODL_2613 EFW95760.1 320581540 Ogataea parapolymorpha DL-1(Hansenula polymorpha DL-1) AAG12171.2 18497328 Mycobacter sp. strainJC1 DSM 3803

FIG. 1, Step Z—Fructose-6-Phosphate Aldolase

Fructose-6-phosphate aldolase (F6P aldolase) can catalyze thecombination of dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate(G3P) to form fructose-6-phosphate. This activity was recentlydiscovered in E. coli and the corresponding gene candidate has beentermed fsa. The enzyme prefers the aldol formation over the cleavagereaction.

The selectivity of the E. coli enzyme towards DHA can be improved byintroducing point mutations. For example, the mutation A129S improvedreactivity towards DHA by over 17 fold in terms of K_(cat)/K_(m)(Gutierrez et al., Chem Commun (Carob), 2011, 47(20), 5762-5764). Genessimilar to fsa have been found in other genomes by sequence homology.Some exemplary gene candidates have been listed below.

Gene Protein accession no. GI number Organism fsa AAC73912.2 87081788Escherichia coli K12 talC AAC76928.1 1790382 Escherichia coli K12 fsaWP_017209835.1 515777235 Clostridium beijerinckii DR_1337 AAF10909.16459090 Deinococcus radiodurans R1 talC NP_213080.1 15605703 Aquifexaeolicus VF5 MJ 0960 NP_247955.1 15669150 Methanocaldococcus janaschiimipB NP_993370.2 161511381 Yersinia pestis

As Described Below, there is an Energetic Advantage to Using F6PAldolase in the DHA Pathway.

The assimilation of formaldehyde formed by the oxidation of methanol canproceed either via the dihydroxyacetone (DHA) pathway (step D, FIG. 1)or the Ribulose monophosphate (RuMP) pathway (steps B and C, FIG. 1). Inthe RuMP pathway, formaldehyde combines with ribulose-5-phosphate toform F6P. F6P is then either metabolized via glycolysis or used forregeneration of ribulose-5-phosphate to enable further formaldehydeassimilation. Notably, ATP hydrolysis is not required to form F6P fromformaldehyde and ribulose-5-phosphate via the RuMP pathway.

In contrast, in the DHA pathway, formaldehyde combines withxylulose-5-phosphate (X5P) to form dihydroxyacetone (DHA) andglyceraldehyde-3-phosphate (G3P). Some of the DHA and G3P must bemetabolized to F6P to enable regeneration of xylulose-5-phosphate. Inthe standard DHA pathway, DHA and G3P are converted to F6P by threeenzymes: DHA kinase, fructose bisphosphate aldolase, and fructosebisphosphatase. The net conversion of DHA and G3P to F6P requires ATPhydrolysis as described below. First, DHA is phosphorylated to form DHAphosphate (DHAP) by DHA kinase at the expense of an ATP. DHAP and G3Pare then combined by fructose bisphosphate aldolase to formfructose-1,6-diphosphate (FDP). FDP is converted to F6P by fructosebisphosphatase, thus wasting a high energy phosphate bond.

A more ATP efficient sequence of reactions is enabled if DHA synthasefunctions in combination with F6P aldolase as opposed to in combinationwith DHA kinase, fructose bisphosphate aldolase, and fructosebisphosphatase. F6P aldolase enables direct conversion of DHA and G3P toF6P, bypassing the need for ATP hydrolysis. Overall, DHA synthase whencombined with F6P aldolase is identical in energy demand to the RuMPpathway. Both of these formaldehyde assimilation options (i.e., RuMPpathway, DHA synthase+F6P aldolase) are superior to DHA synthasecombined with DHA kinase, fructose bisphosphate aldolase, and fructosebisphosphatase in terms of ATP demand.

Example IV In Vivo Labeling Assay for Conversion of Methanol to CO₂

This example describes a functional methanol pathway in a microbialorganism.

Strains with functional reductive TCA branch and pyruvate formate lyasedeletion were grown aerobically in LB medium overnight, followed byinoculation of M9 high-seed media containing IPTG and aerobic growth for4 hrs. These strains had methanol dehydrogenase/ACT pairs in thepresence and absence of formaldehyde dehydrogenase or formatedehydrogenase. ACT is an activator protein (a Nudix hydrolase). At thistime, strains were pelleted, resuspended in fresh M9 medium high-seedmedia containing 2% ¹³CH₃OH, and sealed in anaerobic vials. Head spacewas replaced with nitrogen and strains grown for 40 hours at 37° C.Following growth, headspace was analyzed for ¹³CO₂. Media was examinedfor residual methanol as well as 1,4-butanediol and byproducts. Allconstructs expressing methanol dehydrogenase (MeDH) mutants and MeDH/ACTpairs grew to slightly lower ODs than strains containing empty vectorcontrols. This is likely due to the high expression of these constructs(Data not shown). One construct (2315/2317) displayed significantaccumulation of labeled CO₂ relative to controls in the presence ofFalDH, FDH or no coexpressed protein. This shows a functional MeOHpathway in E. coli and that the endogenous glutathione-dependentformaldehyde detoxification genes (frmAB) are sufficient to carry fluxgenerated by the current MeDH/ACT constructs.

2315 is internal laboratory designation for the MeDH from Bacillusmethanolicus MGA3 (GenBank Accession number EIJ77596.1; GI number387585261), and 2317 is internal laboratory designation for theactivator protein from the same organism (locus tag: MGA3_09170; GenBankAccession number EIJ83380; GI number: 387591061).

Sequence analysis of the NADH-dependent MeDH from Bacillus methanolicusplaces the enzyme in the alcohol dehydrogenase family III. It does notcontain any tryptophan residues, resulting in a low extinctioncoefficient (18,500 M⁻¹, cm⁻¹) and should be detected on SDS gels byCoomassie staining.

The enzyme has been characterized as a multisubunit complex built from43 kDa subunits containing one Zn and 1-2 Mg atoms per subunit. Electronmicroscopy and sedimentation studies determined it to be a decamer, inwhich two rings with five-fold symmetry are stacked on top of each other(Vonck et al., J. Biol. Chem. 266:3949-3954, 1991). It is described tocontain a tightly but not covalently bound cofactor and requiresexogenous NAD⁺ as e⁻-acceptor to measure activity in vitro. A strongincrease (10-40-fold) of in vitro activity was observed in the presenceof an activator protein (ACT), which is a homodimer (21 kDa subunits)and contains one Zn and one Mg atom per subunit.

The mechanism of the activation was investigated by Kloosterman et al.,J. Biol. Chem. 277:34785-34792, 2002, showing that ACT is a Nudixhydrolase and Hektor et al., J. Biol. Chem. 277:46966-46973, 2002,demonstrating that mutation of residue S97 to G or Tin MeDH changesactivation characteristics along with the affinity for the cofactor.While mutation of residues G15 and D88 had no significant impact, a roleof residue G13 for stability as well as of residues G95, D100, and K103for the activity is suggested. Both papers together propose a hypothesisin which ACT cleaves MeDH-bound NAD⁺. MeDH retains AMP bound and entersan activated cycle with increased turnover. The stoichiometric ratiobetween ACT and MeDH is not well defined in the literature. Kloostermanet al., supra determine the ratio of dimeric Act to decameric MeDH forfull in vitro activation to be 10:1. In contrast, Arfman et al. J. Biol.Chem. 266:3955-3960, 1991 determined a ratio of 3:1 in vitro for maximumand a 1:6 ratio for significant activation, but observe a highsensitivity to dilution. Based on expression of both proteins inBacillus, the authors estimate the ratio in vivo to be around 1:17.5.

However, our in vitro experiments with purified activator protein(2317A) and methanol dehydrogenase (2315A) showed the ratio of ACT toMeDH to be 10:1. This in vitro test was done with 5 M methanol, 2 mM NADand 10 μM methanol dehydrogenase 2315A at pH 7.4.

Example V Phosphoketolase-Dependent Acetyl-CoA Synthesis Enzymes

This Example provides genes that can be used for enhancing carbon fluxthrough acetyl-CoA using phosphoketolase enzymes.

FIG. 1, Step T—Fructose-6-Phosphate Phosphoketolase

Conversion of fructose-6-phosphate and phosphate to acetyl-phosphate anderythrose-5-phosphate can be carried out by fructose-6-phosphatephosphoketolase (EC 4.1.2.22). Conversion of fructose-6-phosphate andphosphate to acetyl-phosphate and erythrose-5-phosphate is one of thekey reactions in the Bifidobacterium shunt. There is evidence for theexistence of two distinct phosphoketolase enzymes in bifidobacteria(Sgorbati et al, 1976, Antonie Van Leeuwenhoek, 42(1-2) 49-57; Grill etal, 1995, Curr Microbiol, 31(1); 49-54). The enzyme from Bifidobacteriumdentium appeared to be specific solely for fructose-6-phosphate (EC:4.1.2.22) while the enzyme from Bifidobacterium pseudolongum subsp.globosum is able to utilize both fructose-6-phosphate and D-xylulose5-phosphate (EC: 4.1.2.9) (Sgorbati et al, 1976, Antonie VanLeeuwenhoek, 42(1-2) 49-57). The enzyme encoded by the xfp gene,originally discovered in Bifidobacterium animalis lactis, is thedual-specificity enzyme (Meile et al., 2001, J Bacteriol, 183,2929-2936; Yin et al, 2005, FEMS Microbiol Lett, 246(2); 251-257).Additional phosphoketolase enzymes can be found in Leuconostocmesenteroides (Lee et al, Biotechnol Lett. 2005 June; 27(12):853-8),Clostridium acetobutylicum ATCC 824 (Servinsky et al, Journal ofIndustrial Microbiology & Biotechnology, 2012, 39, 1859-1867),Aspergillus nidulans (Kocharin et al, 2013, Biotechnol Bioeng, 110(8),2216-2224; Papini, 2012, Appl Microbiol Biotechnol, 95 (4), 1001-1010),Bifidobacterium breve (Suziki et al, 2010, Acta Crystallogr Sect FStruct Biol Cryst Commun., 66(Pt 8):941-3), Lactobacillus paraplantarum(Jeong et al, 2007, J Microbiol Biotechnol, 17(5), 822-9).

Protein GENBANK ID GI NUMBER ORGANISM xfp YP_006280131.1 386867137Bifidobacterium animalis lactis xfp AAV66077.1 55818565 Leuconostocmesenteroides CAC1343 NP_347971.1 15894622 Clostridium acetobutylicumATCC 824 xpkA CBF76492.1 259482219 Aspergillus nidulans xfpWP_003840380.1 489937073 Bifidobacterium dentium ATCC 27678 xfpAAR98788.1 41056827 Bifidobacterium pseudolongum subsp. globosum xfpWP_022857642.1 551237197 Bifidobacterium pseudolongum subsp. globosumxfp ADF97524.1 295314695 Bifidobacterium breve xfp AAQ64626.1 34333987Lactobacillus paraplantarum

FIG. 1, Step U—Xylulose-5-Phosphate Phosphoketolase

Conversion of xylulose-5-phosphate and phosphate to acetyl-phosphate andglyceraldehyde-3-phosphate can be carried out by xylulose-5-phosphatephosphoketolase (EC 4.1.2.9). There is evidence for the existence of twodistinct phosphoketolase enzymes in bifidobacteria (Sgorbati et al,1976, Antonie Van Leeuwenhoek, 42(1-2) 49-57; Grill et al, 1995, CurrMicrobiol, 31(1); 49-54). The enzyme from Bifidobacterium dentiumappeared to be specific solely for fructose-6-phosphate (EC: 4.1.2.22)while the enzyme from Bifidobacterium pseudolongum subsp. globosum isable to utilize both fructose-6-phosphate and D-xylulose 5-phosphate(EC: 4.1.2.9) (Sgorbati et al, 1976, Antonie Van Leeuwenhoek, 42(1-2)49-57). Many characterized enzymes have dual-specificity forxylulose-5-phosphate and fructose-6-phosphate. The enzyme encoded by thexfp gene, originally discovered in Bifidobacterium animalis lactis, isthe dual-specificity enzyme (Meile et al., 2001, J Bacteriol, 183,2929-2936; Yin et al, 2005, FEMS Microbiol Lett, 246(2); 251-257).Additional phosphoketolase enzymes can be found in Leuconostocmesenteroides (Lee et al, Biotechnol Let 2005 June; 27(12):853-8),Clostridium acetobutylicum ATCC 824 (Servinsky et al, Journal ofIndustrial Microbiology & Biotechnology, 2012, 39, 1859-1867),Aspergillus nidulans (Kocharin et al, 2013, Biotechnol Bioeng, 110(8),2216-2224; Papini, 2012, Appl Microbiol Biotechnol, 95 (4), 1001-1010),Bifidobacterium breve (Suziki et al, 2010, Acta Crystallogr Sect FStruct Biol Cryst Commun., 66(Pt 8):941-3), and Lactobacillusparaplantarum (Jeong et al, 2007, J Microbiol Biotechnol, 17(5), 822-9).

Protein GENBANK ID GI NUMBER ORGANISM xfp YP_006280131.1 386867137Bifidobacterium animalis lactis xfp AAV66077.1 55818565 Leuconostocmesenteroides CAC1343 NP_347971.1 15894622 Clostridium acetobutylicumATCC 824 xpkA CBF76492.1 259482219 Aspergillus nidulans xfp AAR98788.141056827 Bifidobacterium pseudolongum subsp. globosum xfp WP_022857642.1551237197 Bifidobacterium pseudolongum subsp. globosum xfp ADF97524.1295314695 Bifidobacterium breve xfp AAQ64626.1 34333987 Lactobacillusparaplantarum

FIG. 1, Step V—Phosphotransacetylase

The formation of acetyl-CoA from acetyl-phosphate can be catalyzed byphosphotransacetylase (EC 2.3.1.8). The pta gene from E. coli encodes anenzyme that reversibly converts acetyl-CoA into acetyl-phosphate(Suzuki, T., Biochim. Biophys. Acta 191:559-569 (969)). Additionalacetyltransferase enzymes have been characterized in Bacillus subtilis(Rado and Hoch, Biochim. Biophys. Acta 321:114-125 (1973), Clostridiumkluyveri (Stadtman, E., Methods Enzymol. 1:5896-599 (1955), andThermotoga maritima (Bock et al., J. Bacteriol. 181:1861-1867 (1999)).This reaction can also be catalyzed by some phosphotransbutyrylaseenzymes (EC 2.3.1.19), including the ptb gene products from Clostridiumacetobutylicum (Wiesenbom et al., App. Environ. Microbiol. 55:317-322(1989); Walter et al., Gene 134:107-111 (1993)). Additional ptb genesare found in butyrate-producing bacterium L2-50 (Louis et al., J.Bacteriol. 186:2099-2106 (2004) and Bacillus megaterium (Vazquez et al.,Curr. Microbiol. 42:345-349 (2001). Homologs to the E. coli pta geneexist in several other organisms including Salmonella enterica andChlamydomonas reinhardtii.

Protein GenBank ID GI Number Organism Pta NP_416800.1 71152910Escherichia coli Pta P39646 730415 Bacillus subtilis Pta A5N801146346896 Clostridium kluyveri Pta Q9X0L4 6685776 Thermotoga maritimePtb NP_349676 34540484 Clostridium acetobutylicum Ptb AAR19757.138425288 butyrate-producing bacterium L2-50 Ptb CAC07932.1 10046659Bacillus megaterium Pta NP_461280.1 16765665 Salmonella enterica subsp.enterica serovar Typhimurium str. LT2 PAT2 XP_001694504.1 159472743Chlamydomonas reinhardtii PAT1 XP_001691787.1 159467202 Chlamydomonasreinhardtii

FIG. 1, Step W—Acetate Kinase

Acetate kinase (EC 2.7.2.1) can catalyze the reversible ATP-dependentphosphorylation of acetate to acetylphosphate. Exemplary acetate kinaseenzymes have been characterized in many organisms including E. coli,Clostridium acetobutylicum and Methanosarcina thermophila (Ingram-Smithet al., J. Bacteriol. 187:2386-2394 (2005); Fox and Roseman, J. Biol.Chem. 261:13487-13497 (1986); Winzer et al., Microbioloy 143 (Pt10):3279-3286 (1997)). Acetate kinase activity has also beendemonstrated in the gene product of E. coli purT (Marolewski et al.,Biochemistry 33:2531-2537 (1994). Some butyrate kinase enzymes (EC2.7.2.7), for example buk1 and buk2 from Clostridium acetobutylicum,also accept acetate as a substrate (Hartmanis, M G., J. Biol. Chem.262:617-621 (1987)). Homologs exist in several other organisms includingSalmonella enterica and Chlamydomonas reinhardtii.

Protein GenBank ID GI Number Organism ackA NP_416799.1 16130231Escherichia coli Ack AAB18301.1 1491790 Clostridium acetobutylicum AckAAA72042.1 349834 Methanosarcina thermophila purT AAC74919.1 1788155Escherichia coli buk1 NP_349675 15896326 Clostridium acetobutylicum buk2Q97II1 20137415 Clostridium acetobutylicum ackA NP_461279.1 16765664Salmonella typhimurium ACK1 XP_001694505.1 159472745 Chlamydomonasreinhardtii ACK2 XP_001691682.1 159466992 Chlamydomonas reinhardtii

FIG. 1, Step X—Acetyl-CoA Transferase, Synthetase, or Ligase

The acylation of acetate to acetyl-CoA can be catalyzed by enzymes withacetyl-CoA synthetase, ligase or transferase activity. Two enzymes thatcan catalyze this reaction are AMP-forming acetyl-CoA synthetase orligase (EC 6.2.1.1) and ADP-forming acetyl-CoA synthetase (EC 6.2.1.13).AMP-forming acetyl-CoA synthetase (ACS) is the predominant enzyme foractivation of acetate to acetyl-CoA. ADP-forming acetyl-CoA synthetasesare reversible enzymes with a generally broad substrate range (Musfeldtand Schonheit, J. Bacteriol. 184:636-644 (2002)). The aforementionedproteins are shown below.

Protein GenBank ID GI Number Organism Acs AAC77039.1 1790505 Escherichiacoli acoE AAA21945.1 141890 Ralstonia eutropha acs1 ABC87079.1 86169671Methanothermobacter thermautotrophicus acs1 AAL23099.1 16422835Salmonella enterica ACS1 Q01574.2 257050994 Saccharomyces cerevisiaeAF1211 NP_070039.1 11498810 Archaeoglobus fulgidus AF1983 NP_070807.111499565 Archaeoglobus fulgidus Scs YP_135572.1 55377722 Haloarculamarismortui PAE3250 NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2sucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949Escherichia coli paaF AAC24333.2 22711873 Pseudomonas putida

An acetyl-CoA transferase that can utilize acetate as the CoA acceptoris acetoacetyl-CoA transferase, encoded by the E. coli atoA (alphasubunit) and atoD (beta subunit) genes. This and other proteins areidentified below.

Protein GenBank ID GI Number Organism atoA P76459.1 2492994 Escherichiacoli K12 atoD P76458.1 2492990 Escherichia coli K12 actA YP_226809.162391407 Corynebacterium glutamicum ATCC 13032 cg0592 YP_224801.162389399 Corynebacterium glutamicum ATCC 13032 ctfA NP_149326.1 15004866Clostridium acetobutylicum ctfB NP_149327.1 15004867 Clostridiumacetobutylicum ctfA AAP42564.1 31075384 Clostridiumsaccharoperbutylacetonicum ctfB AAP42565.1 31075385 Clostridiumsaccharoperbutylacetonicum

Additional exemplary acetyl-CoA transferase candidates are catalyzed bythe gene products of cat1, cat2, and cat3 of Clostridium kluyveri whichhave been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, andbutyryl-CoA transferase activity, respectively. Similar CoA transferaseactivities are also present in Trichomonas vaginalis (van Grinsven etal., J. Biol. Chem. 283:1411-1418 (2008)) and Trypanosoma brucei(Riviere et al., J. Biol. Chem. 279:45337-45346 (2004)). These and otherproteins are identified below.

Protein GenBank ID GI Number Organism cat1 P38946.1 729048 Clostridiumkluyveri cat2 P38942.2 172046066 Clostridium kluyveri cat3 EDK35586.1146349050 Clostridium kluyveri TVAG_395550 XP_001330176 123975034Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875 Trypanosomabrucei

Example VI Acetyl-CoA and Succinyl-CoA Synthesis Enzymes

This Example provides genes that can be used for conversion ofglycolysis intermediate glyceraldehyde-3-phosphate (G3P) to acetyl-CoAand/or succinyl-CoA as depicted in the pathways of FIG. 4.

A. PEP Carboxylase or PEP Carboxykinase.

Carboxylation of phosphoenolpyruvate to oxaloacetate is catalyzed byphosphoenolpyruvate carboxylase. Exemplary PEP carboxylase enzymes arebelow.

Protein GenBank ID GI Number Organism Ppc NP_418391 16131794 Escherichiacoli ppcA AAB58883 28572162 Methylobacterium extorquens Ppc ABB5327080973080 Corynebacterium glutamicum

An alternative enzyme for converting phosphoenolpyruvate to oxaloacetateis PEP carboxykinase, which simultaneously forms an ATP whilecarboxylating PEP. In most organisms PEP carboxykinase serves agluconeogenic function and converts oxaloacetate to PEP at the expenseof one ATP. S. cerevisiae is one such organism whose native PEPcarboxykinase, PCK1, serves a gluconeogenic role (Valdes-Hevia et al.,FEBS Lett. 258:313-316 (1989). E. coli is another such organism, as therole of PEP carboxykinase in producing oxaloacetate is believed to beminor when compared to PEP carboxylase, which does not form ATP,possibly due to the higher K_(m) for bicarbonate of PEP carboxykinase(Kim et al., Appl. Environ. Microbiol. 70:1238-1241 (2004)).Nevertheless, activity of the native E. coli PEP carboxykinase from PEPtowards oxaloacetate has been recently demonstrated in ppc mutants of E.coli K-12 (Kwon et al., J. Microbiol. Biotechnol. 16:1448-1452 (2006)).These strains exhibited no growth defects and had increased succinateproduction at high NaHCO₃ concentrations. Mutant strains of E. coli canadopt Pck as the dominant CO2-fixing enzyme following adaptive evolution(Zhang et al. 2009). In some organisms, particularly rumen bacteria, PEPcarboxykinase is quite efficient in producing oxaloacetate from PEP andgenerating ATP. Examples of PEP carboxykinase genes are shown below.

Protein GenBank ID GI Number Organism PCK1 NP_013023 6322950Saccharomyces cerevisiae pck NP_417862.1 16131280 Escherichia coli pckAYP_089485.1 52426348 Mannheimia succiniciproducens pckA O09460.1 3122621Anaerobiospirillum succiniciproducens pckA Q6W6X5 75440571Actinobacillus succinogenes pckA P43923.1 1172573 Haemophilus influenza

B. Malate Dehydrogenase.

Oxaloacetate is converted into malate by malate dehydrogenase (EC1.1.1.37), an enzyme which functions in both the forward and reversedirection. Exemplary enzymes are show below.

Protein GenBank ID GI Number Organism MDH1 NP_012838 6322765Saccharomyces cerevisiae MDH2 NP_014515 116006499 Saccharomycescerevisiae MDH3 NP_010205 6320125 Saccharomyces cerevisiae MdhNP_417703.1 16131126 Escherichia coli

C. Fumarase.

Fumarate hydratase (EC 4.2.1.2) catalyzes the reversible hydration offumarate to malate. The three fumarases of E. coli, encoded by fumA,fumB and fumC, are regulated under different conditions of oxygenavailability. FumB is oxygen sensitive and is active under anaerobicconditions. FumA is active under microanaerobic conditions, and FumC isactive under aerobic growth conditions (Tseng et al., J. Bacteriol.183:461-467 (2001); Woods et al., Biochim. Biophys. Acta 954:14-26(1988); Guest et al., J. Gen. Microbiol. 131:2971-2984 (1985)).Additional fumarase enzymes are shown below.

Protein GenBank ID GI Number Organism fumA NP_416129.1 16129570Escherichia coli fumB NP_418546.1 16131948 Escherichia coli fumCNP_416128.1 16129569 Escherichia coli FUM1 NP_015061 6324993Saccharomyces cerevisiae fumC Q8NRN8.1 39931596 Corynebacteriumglutamicum fumC O69294.1 9789756 Campylobacter jejuni fumC P8412775427690 Thermus thermophilus fumH P14408.1 120605 Rattus norvegicusMmcB YP_001211906 147677691 Pelotomaculum thermopropionicum MmcCYP_001211907 147677692 Pelotomaculum thermopropionicum

D. Fumarate Reductase.

Fumarate reductase catalyzes the reduction of fumarate to succinate. Thefumarate reductase of E. coli, composed of four subunits encoded byfrdABCD, is membrane-bound and active under anaerobic conditions. Theelectron donor for this reaction is menaquinone and the two protonsproduced in this reaction do not contribute to the proton gradient(Iverson et al., Science 284:1961-1966 (1999)). The yeast genome encodestwo soluble fumarate reductase isozymes encoded by FRDS1 and FRDS2.

Protein GenBank ID GI Number Organism FRDS1 P32614 418423 Saccharomycescerevisiae FRDS2 NP_012585 6322511 Saccharomyces cerevisiae frdANP_418578.1 16131979 Escherichia coli frdB NP_418577.1 16131978Escherichia coli frdC NP_418576.1 16131977 Escherichia coli frdDNP_418475.1 16131877 Escherichia coli

E. Succinyl-CoA Synthetase or Transferase.

The ATP-dependent acylation of succinate to succinyl-CoA is catalyzed bysuccinyl-CoA synthetase (EC 6.2.1.5). The product of the LSC1 and LSC2genes of S. cerevisiae and the sucC and sucD genes of E. coli naturallyform a succinyl-CoA synthetase complex that catalyzes the formation ofsuccinyl-CoA from succinate with the concomitant consumption of one ATP,a reaction which is reversible in vivo. These proteins are identifiedbelow:

Protein GenBank ID GI Number Organism LSC1 NP_014785 6324716Saccharomyces cerevisiae LSC2 NP_011760 6321683 Saccharomyces cerevisiaesucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949Escherichia coli

Succinyl-CoA transferase converts succinate and an acyl-CoA donor tosuccinyl-CoA and an acid. Succinyl-CoA transferase enzymes include ygfHof E. coli, cat1 of Clostridium kluyveri, and other exemplary enzymesshown below. Additional CoA transferases, described herein, are alsosuitable candidates.

Gene GI # Accession No. Organism ygfH AAC75957.1 1789287 Escherichiacoli cat1 P38946.1 729048 Clostridium kluyveri CIT292_04485ZP_03838384.1 227334728 Citrobacter youngae SARI 04582 YP_001573497.1161506385 Salmonella enterica yinte0001_14430 ZP_04635364.1 238791727Yersinia intermedia pcaI 24985644 AAN69545.1 Pseudomonas putida pcaJ26990657 NP_746082.1 Pseudomonas putida pcaI 50084858 YP_046368.1Acinetobacter sp. ADP1 pcaJ 141776 AAC37147.1 Acinetobacter sp. ADP1pcaI 21224997 NP_630776.1 Streptomyces coelicolor pcaJ 21224996NP_630775.1 Streptomyces coelicolor catI 75404583 Q8VPF3 Pseudomonasknackmussii catJ 75404582 Q8VPF2 Pseudomonas knackmussii HPAG1 0676108563101 YP_627417 Helicobacter pylori HPAG1_0677 108563102 YP_627418Helicobacter pylori ScoA 16080950 NP_391778 Bacillus subtilis ScoB16080949 NP_391777 Bacillus subtilis OXCT1 NP_000427 4557817 Homosapiens OXCT2 NP_071403 11545841 Homo sapiens

F. Pyruvate Kinase or PTS-Dependent Substrate Import. See ElsewhereHerein.

G. Pyruvate Dehydrogenase, Pyruvate Formate Lyase or Pyruvate:FerredoxinOxidoreductase.

Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the reversibleoxidation of pyruvate to form acetyl-CoA. Exemplary PFOR enzymes arefound in Desulfovibrio africanus (Pieulle et al., J. Bacteriol.179:5684-5692 (1997)) and other Desulfovibrio species (Vita et al.,Biochemistry, 47: 957-64 (2008)). The M. thermoacetica PFOR is also wellcharacterized (Menon and Ragsdale, Biochemistry 36:8484-8494 (1997)) andwas shown to have high activity in the direction of pyruvate synthesisduring autotrophic growth (Furdui and Ragsdale, J. Biol. Chem.275:28494-28499 (2000)). Further, E. coli possesses an uncharacterizedopen reading frame, ydbK, encoding a protein that is 51% identical tothe M. thermoacetica PFOR. Evidence for pyruvate oxidoreductase activityin E. coli has been described (Blaschkowski et al., Eur. J. Biochem.123:563-569 (1982)). Finally, flavodoxin reductases (e.g., fqrB fromHelicobacter pylori or Campylobacter jejuni) (St Maurice et al., J.Bacteriol. 189:4764-4773 (2007)) or Rnf-type proteins (Seedorf et al.,Proc. Natl. Acad. Sci. USA. 105:2128-2133 (2008); and Hellmann, J.Bacteriol 190:784-791 (2008)) provide a means to generate NADH or NADPHfrom the reduced ferredoxin generated by PFOR

Protein GenBank ID GI Number Organism DesfrDRAFT_0121 ZP_07331646.1303245362 Desulfovibrio fructosovorans JJ Por CAA70873.1 1770208Desulfovibrio africanus por YP_012236.1 46581428 Desulfovibrio vulgarisstr. Hildenborough Dde_3237 ABB40031.1 78220682 DesulfoVibriodesulfuricans G20 Ddes_0298 YP_002478891.1 220903579 Desulfovibriodesulfuricans subsp. desulfuricans str. ATCC 27774 Por YP_428946.183588937 Moorella thermoacetica YdbK NP_415896.1 16129339 Escherichiacoli

The conversion of pyruvate into acetyl-CoA can be catalyzed by severalother enzymes or their combinations thereof. For example, pyruvatedehydrogenase can transform pyruvate into acetyl-CoA with theconcomitant reduction of a molecule of NAD into NADH. It is amulti-enzyme complex that catalyzes a series of partial reactions whichresults in acylating oxidative decarboxylation of pyruvate. The enzymecomprises of three subunits: pyruvate decarboxylase (E1),dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase(E3). This enzyme is naturally present in several organisms, includingE. coli and S. cerevisiae. Enzyme engineering efforts have improved theE. coli PDH enzyme activity under anaerobic conditions.

Gene Accession No. GI # Organism aceE NP_414656.1 16128107 Escherichiacoli aceF NP_414657.1 16128108 Escherichia coli lpd NP_414658.1 16128109Escherichia coli pdhA P21881.1 3123238 Bacillus subtilis pdhB P21882.1129068 Bacillus subtilis pdhC P21883.2 129054 Bacillus subtilis pdhDP21880.1 118672 Bacillus subtilis LAT1 NP_014328 6324258 Saccharomycescerevisiae PDA1 NP_011105 37362644 Saccharomyces cerevisiae PDB1NP_009780 6319698 Saccharomyces cerevisiae LPD1 NP_116635 14318501Saccharomyces cerevisiae PDX1 NP_011709 6321632 Saccharomyces cerevisiae

Yet another enzyme that can catalyze this conversion is pyruvate formatelyase. This enzyme catalyzes the conversion of pyruvate and CoA intoacetyl-CoA and formate. Pyruvate formate lyase is a common enzyme inprokaryotic organisms that is used to help modulate anaerobic redoxbalance. Exemplary enzymes are below. Both pflB and tdcE from E. colirequire the presence of pyruvate formate lyase activating enzyme,encoded by pflA. Further, a short protein encoded by yflD in E. coli canassociate with and restore activity to oxygen-cleaved pyruvate formatelyase.

Protein GenBank ID GI Number Organism pflB NP_415423 16128870Escherichia coli pflA NP_415422.1 16128869 Escherichia coli tdcEAAT48170.1 48994926 Escherichia coli yfiD AAC75632.1 1788933 Escherichiacoli pfl Q46266.1 2500058 Clostridium pasteurianum act CAA63749.11072362 Clostridium pasteurianum

Further, different enzymes can be used in combination to convertpyruvate into acetyl-CoA in multiple steps. For example, in S.cerevisiae, acetyl-CoA is obtained in the cytosol by firstdecarboxylating pyruvate to form acetaldehyde; the latter is oxidized toacetate by acetaldehyde dehydrogenase and subsequently activated to formacetyl-CoA by acetyl-CoA synthetase. Acetyl-CoA synthetase is a nativeenzyme in several other organisms including E. coli (Kumari et al., J.Bacteriol. 177:2878-2886 (1995)), Salmonella enterica (Starai et al.,Microbiology 151:3793-3801 (2005); Starai et al., J. Biol. Chem.280:26200-26205 (2005)), and Moorella thermoacetica (described already).Alternatively, acetate can be activated to form acetyl-CoA by acetatekinase and phosphotransacetylase. Acetate kinase first converts acetateinto acetyl-phosphate with the accompanying use of an ATP molecule.Acetyl-phosphate and CoA are next converted into acetyl-CoA with therelease of one phosphate by phosphotransacetylase. Exemplary enzymesencoding acetate kinase, acetyl-CoA synthetase and phosphotransacetlyaseare described above.

Yet another way of converting pyruvate to acetyl-CoA is via pyruvateoxidase. Pyruvate oxidase converts pyruvate into acetate, usingubiquinone as the electron acceptor. In E. coli, this activity isencoded by poxB. PoxB has similarity to pyruvate decarboxylase of S.cerevisiae and Zymomonas mobilis. The enzyme has a thiamin pyrophosphatecofactor (Koland and Gennis, Biochemistry 21:4438-4442 (1982)); O'Brienet al., Biochemistry 16:3105-3109 (1977); O'Brien and Gennis, J. Biol.Chem. 255:3302-3307 (1980)) and a flavin adenine dinucleotide (FAD)cofactor. Acetate can then be converted into acetyl-CoA by eitheracetyl-CoA synthetase or by acetate kinase and phosphotransacetylase, asdescribed earlier. Some of these enzymes can also catalyze the reversereaction from acetyl-CoA to pyruvate.

H. Citrate Synthase.

Citrate synthases are well known in the art. For exampe, the gltA geneof E. coli encodes for a citrate synthase. It was previously shown thatthis gene is inhibited allosterically by NADH, and the amino acidsinvolved in this inhibition have been identified (Pereira et al., J.Biol. Chem. 269(1):412-417 (1994); Stokell et al., J. Biol. Chem.278(37):35435-35443 (2003)). An NADH insensitive citrate synthase can beencoded by gltA, such as an R163L mutant of gltA. Other citrate synthaseenzymes are less sensitive to NADH, including the aarA enzyme ofAcetobacter aceti (Francois et al, Biochem 45:13487-99 (2006)).

Protein GenBank ID GI number Organism gltA NP_415248.1 16128695Escherichia coli AarA P20901.1 116462 Acetobacter aceti CITI NP_014398.16324328 Saccharomyces cerevisiae CS NP_999441.1 47523618 Sus scrofa

I. Aconitase.

Aconitase (EC 4.2.1.3) is an iron-sulfur-containing protein catalyzingthe reversible isomerization of citrate and iso-citrate via theintermediate cis-aconitate. Two aconitase enzymes of E. coli are encodedby acnA and acnB. AcnB is the main catabolic enzyme, while AcnA is morestable and appears to be active under conditions of oxidative or acidstress. Exemplary enzymes are below.

Protein GenBank ID GI Number Organism acnA AAC7438.1 1787531 Escherichiacoli acnB AAC73229.1 2367097 Escherichia coli HP0779 NP_207572.115645398 Helicobacter pylori 26695 H16_B0568 CAJ95365.1 113529018Ralstonia eutropha DesfrDRAFT_3783 ZP_07335307.1 303249064 Desulfovibriofructosovorans JJ Suden_1040 (acnB) ABB44318.1 78497778 Sulfurimonasdenitrificans Hydth_0755 ADO45152.1 308751669 Hydrogenobacterthermophilus CT0543 (acn) AAM71785.1 21646475 Chlorobium tepidumClim_2436 YP_001944436.1 189347907 Chlorobium limicola Clim_0515ACD89607.1 189340204 Chlorobium limicola acnA NP_460671.1 16765056Salmonella typhimurium acnB NP_459163.1 16763548 Salmonella typhimuriumACO1 AAA34389.1 170982 Saccharomyces cerevisiae

J. Isocitrate Dehydrogenase.

Isocitrate dehydrogenase catalyzes the decarboxylation of isocitrate to2-oxoglutarate coupled to the reduction of NAD(P)⁺. Exemplary enzymesare listed below.

Protein GenBank ID GI Number Organism Icd ACI84720.1 209772816Escherichia coli IDP1 AAA34703.1 171749 Saccharomyces cerevisiae IdhBAC00856.1 21396513 Chlorobium limicola Icd AAM71597.1 21646271Chlorobium tepidum icd NP_952516.1 39996565 Geobacter sulfurreducens icdYP_393560. 78777245 Sulfurimonas denitrificans

K. AKG Dehydrogenase.

Alpha-ketoglutarate dehydrogenase (AKGD) converts alpha-ketoglutarate tosuccinyl-CoA and is the primary site of control of metabolic fluxthrough the TCA cycle (Hansford, Curr. Top. Bioenerg. 10:217-278(1980)). Exemplary AKGDH enzymes are listed below.

Gene GI # Accession No. Organism sucA 16128701 NP_415254.1 Escherichiacoli sucB 16128702 NP_415255.1 Escherichia coli lpd 16128109 NP_414658.1Escherichia coli odhA 51704265 P23129.2 Bacillus subtilis odhB 129041P16263.1 Bacillus subtilis pdhD 118672 P21880.1 Bacillus subtilis KGD16322066 NP_012141.1 Saccharomyces cerevisiae KGD2 6320352 NP_010432.1Saccharomyces cerevisiae LPD1 14318501 NP_116635.1 Saccharomycescerevisiae

The conversion of alpha-ketoglutarate to succinyl-CoA can also becatalyzed by alpha-ketoglutamte:ferredoxin oxidoreductase (EC 1.2.7.3),also known as 2-oxoglutamte synthase or 2-oxoglutamte:ferredoxinoxidoreductase (OFOR). OFOR and pyruvate:ferredoxin oxidoreductase(PFOR) are members of a diverse family of 2-oxoacid:ferredoxin(flavodoxin) oxidoreductases which utilize thiamine pyrophosphate, CoAand iron-sulfur clusters as cofactors and ferredoxin, flavodoxin and FADas electron carriers (Adams et al., Archaea. Adv. Protein Chem.48:101-180 (1996)). Exemplary OFOR enzymes are found in organisms suchas Hydrogenobacter thermophilus, Desulfobacter hydrogenophilus andChlorobium species (Shiba et al. 1985; Evans et al., Proc. Natl. Acad.ScI. USA. 55:92934 (1966); Buchanan, 1971). The two-subunit enzyme fromH. thermophilus, encoded by korAB, has been cloned and expressed in E.coli. A five subunit OFOR from the same organism with strict substratespecificity for succinyl-CoA, encoded by forDABGE, was recentlyidentified and expressed in E. coli. Exemplary OFOR are below.

Protein GenBank ID GI Number Organism korA BAB21494 12583691Hydrogenobacter thermophilus korB BAB21495 12583692 Hydrogenobacterthermophilus forD BAB62132.1 14970994 Hydrogenobacter thermophilus forABAB62133.1 14970995 Hydrogenobacter thermophilus forB BAB62134.114970996 Hydrogenobacter thermophilus forG BAB62135.1 14970997Hydrogenobacter thermophilus forE BAB62136.1 14970998 Hydrogenobacterthermophilus Clim 0204 ACD89303.1 189339900 Chlorobium limicola Clim0205 ACD89302.1 189339899 Chlorobium limicola Clim 1123 ACD90192.1189340789 Chlorobium limicola Clim_1124 ACD90193.1 189340790 Chlorobiumlimicola korA CAA12243.2 19571179 Thauera aromatica korB CAD27440.119571178 Thauera aromatica

L. Pyruvate Carboxylase.

Pyruvate carboxylase (EC 6.4.1.1) directly converts pyruvate tooxaloacetate at the cost of one ATP. Pyruvate carboxylase enzymes arebelow.

Protein GenBank ID GI Number Organism PYC1 NP_011453 6321376Saccharomyces cerevisiae PYC2 NP_009777 6319695 Saccharomyces cerevisiaePyc YP_890857.1 118470447 Mycobacterium smegmatis

M. Malic Enzyme.

Malic enzyme can be applied to convert CO₂ and pyruvate to malate at theexpense of one reducing equivalent. Malic enzymes for this purpose caninclude, without limitation, malic enzyme (NAD-dependent) and malicenzyme (NADP-dependent). For example, one of the E. coli malic enzymes(Takeo, I Biochem. 66:379-387 (1969)) or a similar enzyme with higheractivity can be expressed to enable the conversion of pyruvate and CO₂to malate. By fixing carbon to pyruvate as opposed to PEP, malic enzymeallows the high-energy phosphate bond from PEP to be conserved bypyruvate kinase whereby ATP is generated in the formation of pyruvate orby the phosphotransferase system for glucose transport.

Protein GenBank ID GI Number Organism maeA NP_415996 90111281Escherichia coli maeB NP_416958 16130388 Escherichia coli NAD-ME P27443126732 Ascaris suum

Example VII 1,3-Butanediol, Crotyl Alcohol, 3-Buten-2-ol, and ButadieneSynthesis Enzymes

This Example provides genes that can be used for conversion ofacetyl-CoA to 1,3-butanediol, crotyl alcohol, 3-buten-2-ol, butadiene asdepicted in the pathways of FIGS. 5 and 6.

FIG. 5. Pathways for converting 1,3-butanediol to 3-buten-2-ol and/orbutadiene. A) acetyl-CoA carboxylase, B) an acetoacetyl-CoA synthase, C)an acetyl-CoA:acetyl-CoA acyltransferase, D) an acetoacetyl-CoAreductase (ketone reducing), E) a 3-hydroxybutyryl-CoA reductase(aldehyde forming), F) a 3-hydroxybutyryl-CoA hydrolase, transferase orsynthetase, G) a 3-hydroxybutyrate reductase, H) a3-hydroxybutyraldehyde reductase, I) chemical dehydration orcorresponding step in FIG. 6, J) a 3-hydroxybutyryl-CoA dehydratase, K)a crotonyl-CoA reductase (aldehyde forming), L) a crotonyl-CoAhydrolase, transferase or synthetase, M) a crotonate reductase, N) acrotonaldehyde reductase, O) a crotyl alcohol kinase, P) a2-butenyl-4-phosphate kinase, Q) a butadiene synthase, R) a crotylalcohol diphosphokinase, S) chemical dehydration or a crotyl alcoholdehydratase, T) a butadiene synthase (monophosphate), T) a butadienesynthase (monophosphate), U) a crotonyl-CoA reductase (alcohol forming),and V) a 3-hydroxybutyryl-CoA reductase (alcohol forming).

A. Acetyl-CoA Carboxylase.

Acetyl-CoA carboxylase (EC 6.4.1.2) catalyzes the ATP-dependentcarboxylation of acetyl-CoA to malonyl-CoA. This enzyme is biotindependent and is the first reaction of fatty acid biosynthesisinitiation in several organisms. Exemplary enzymes are below.

Protein GenBank ID GI Number Organism ACC1 CAA96294.1 1302498Saccharomyces cerevisiae KLLA0F06072g XP_455355.1 50310667 Kluyveromyceslactis ACC1 XP_718624.1 68474502 Candida albicans YALI0C11407pXP_501721.1 50548503 Yarrowia lipolytica ANI_1_1724104 XP_001395476.1145246454 Aspergillus niger accA AAC73296.1 1786382 Escherichia coliaccB AAC76287.1 1789653 Escherichia coli accC AAC76288.1 1789654Escherichia coli accD AAC75376.1 1788655 Escherichia coli

B. Acetoacetyl-CoA Synthase.

The conversion of malonyl-CoA and acetyl-CoA substrates toacetoacetyl-CoA can be catalyzed by a CoA synthetase in the 2.3.1 familyof enzymes. Several enzymes catalyzing the CoA synthetase activitieshave been described in the literature and represent suitable candidates.

3-Oxoacyl-CoA products such as acetoacetyl-CoA, 3-oxopentanoyl-CoA,3-oxo-5-hydroxypentanoyl-CoA can be synthesized from acyl-CoA andmalonyl-CoA substrates by 3-oxoacyl-CoA synthases. As enzymes in thisclass catalyze an essentially irreversible reaction, they areparticularly useful for metabolic engineering applications foroverproducing metabolites, fuels or chemicals derived from 3-oxoacyl-CoAintermediates such as acetoacetyl-CoA. Acetoacetyl-CoA synthase, forexample, has been heterologously expressed in organisms thatbiosynthesize butanol (Lan et al, PNAS USA (2012)) andpoly-(3-hydroxybutyrate) (Matsumoto et al, Biosci Biotech Biochem,75:364-366 (2011). Other acetoacetyl-CoA synthase genes can beidentified by sequence homology to fhsA

Protein GenBank ID GI Number Organism fhsA BAJ83474.1 325302227Streptomyces sp CL190 AB183750.1: 11991 . . . 12971 BAD86806.1 57753876Streptomyces sp. KO-3988 epzT ADO43379.1 312190954 Strentomvcescinnamonensis ppzT CAX48662.1 238623523 Streptomyces anulatus O3I 22085ZP 09840373.1 378817444 Nocardia brasiliensis

C. Acetyl-CoA:acetyl-CoA Acyltransferase (Acetoacetyl-CoA Thiolase).

Acetoacetyl-CoA thiolase (also known as acetyl-CoA acetyltransferase)converts two molecules of acetyl-CoA into one molecule each ofacetoacetyl-CoA and CoA. Exemplary acetoacetyl-CoA thiolase enzymesinclude genes/proteins identified in the Table below.

Gene GenBank ID GI Number Organism AtoB NP_416728 16130161 Escherichiacoli ThlA NP_349476.1 15896127 Clostridium acetobutylicum ThlBNP_149242.1 15004782 Clostridium acetobutylicum ERG10 NP_015297 6325229Saccharomyces cerevisiae phbA P07097.4 135759 Zoogloea ramigera

D. Acetoacetyl-CoA Reductase.

A suitable enzyme activity is 1.1.1.a Oxidoreductase (oxo to alcohol).See herein. In addition, Acetoacetyl-CoA reductase (EC 1.1.1.36)catalyzes the reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. Thisenzyme participates in the acetyl-CoA fermentation pathway to butyratein several species of Clostridia and has been studied in detail (Joneset al., Microbiol Rev. 50:484-524 (1986)). Acetoacetyl-CoA reductasealso participates in polyhydroxybutyrate biosynthesis in many organisms,and has also been used in metabolic engineering applications foroverproducing PHB and 3-hydroxyisobutyrate. Additional exemplary genesinclude those below. The Z ramigera gene is NADPH-dependent and the genehas been expressed in E coli (Peoples et al., Mol. Microbiol 3:349-357(1989)). Substrate specificity studies on the gene led to the conclusionthat it could accept 3-oxopropionyl-CoA as a substrate besidesacetoacetyl-CoA (Ploux et al., Eur. J Biochem. 174:177-182 (1988)). Theenzyme from Candida tropicalis is a component of the peroxisomal fattyacid beta-oxidation multifunctional enzyme type 2 (MFE-2). Thedehydrogenase B domain of this protein is catalytically active onacetoacetyl-CoA.

Protein Genbank ID GI Number Organism fadB P21177.2 119811 Escherichiacoli fadJ P77399.1 3334437 Escherichia coli paaH NP_415913.1 16129356Escherichia coli Hbd2 EDK34807.1 146348271 Clostridium kluyveri Hbd1EDK32512.1 146345976 Clostridium kluyveri phaC NP_745425.1 26990000Pseudomonas putida paaC ABF82235.1 106636095 Pseudomonas fluorescensHSD17B10 O02691.3 3183024 Bos taurus phbB P23238.1 130017 Zoogloearamigera phaB YP_353825.1 77464321 Rhodobacter sphaeroides phaB BAA08358675524 Paracoccus denitrificans Hbd NP_349314.1 15895965 Clostridiumacetobutylicum Hbd AAM14586.1 20162442 Clostridium beijerinckii Msed1423 YP_001191505 146304189 Metallosphaera sedula Msed_0399 YP_001190500146303184 Metallosphaera sedula Msed 0389 YP_001190490 146303174Metallosphaera sedula Msed_1993 YP_001192057 146304741 Metallosphaerasedula Fox2 Q02207 399508 Candida tropicalis

E. 3-Hydroxybutyryl-CoA Reductase (Aldehyde Forming).

An EC 1.2.1.b Oxidoreductase (acyl-CoA to aldehyde) provides suitableenzyme activity. Acyl-CoA reductases or acylating aldehydedehydrogenases reduce an acyl-CoA to its corresponding aldehyde.Exemplary enzymes include fatty acyl-CoA reductase, succinyl-CoAreductase (EC 1.2.1.76), acetyl-CoA reductase, butyryl-CoA reductase,propionyl-CoA reductase (EC 1.2.1.3) and others shown in the tablebelow.

EC Number Enzyme name 1.2.1.10 Acetaldehyde dehydrogenase (acetylating)1.2.1.42 (Fatty) acyl-CoA reductase 1.2.1.44 Cinnamoyl-CoA reductase1.2.1.50 Long chain fatty acyl-CoA reductase 1.2.1.57 Butanaldehydrogenase 1.2.1.75 Malonate semialdehyde dehydrogenase 1.2.1.76Succinate semialdehyde dehydrogenase 1.2.1.81 Sulfoacetaldehydedehydrogenase 1.2.1. — Propanal dehydrogenase 1.2.1.— Hexanaldehydrogenase 1.2.1.— 4-Hydroxybutyraldehyde dehydrogenase

Exemplary fatty acyl-CoA reductases enzymes are encoded by acr1 ofAcinetobacter calcoaceticus (Reiser, Journal of Bacteriology179:2969-2975 (1997)) and Acinetobacter sp. M-1 (Ishige et al., Appl.Environ. Microbiol. 68:1192-1195 (2002)). Enzymes with succinyl-CoAreductase activity are encoded by sucD of Clostridium kluyveri (Sohling,J. Bacteriol. 178:871-880 (1996)) and sucD of P. gingivalis (Takahashi,J. Bacteriol 182:4704-4710 (2000)). Additional succinyl-CoA reductaseenzymes are exemplified below.

Protein GenBank ID GI Number Organism acr1 YP_047869.1 50086359Acinetobacter calcoaceticus acr1 AAC45217 1684886 Acinetobacter baylyiacr1 BAB85476.1 18857901 Acinetobacter sp. Strain M-1 MSED 0709YP_001190808.1 146303492 Metallosphaera sedula Tneu 0421 ACB39369.1170934108 Thermoproteus neutrophilus sucD P38947.1 172046062 Clostridiumkluyveri sucD NP_904963.1 34540484 Porphyromonas gingivalis bphGBAA03892.1 425213 Pseudomonas sp adhE AAV66076.1 55818563 Leuconostocmesenteroides bld AAP42563.1 31075383 Clostridiumsaccharoperbutylacetonicum pduP NP_460996 16765381 Salmonellatyphimurium LT2 eutE NP_416950 16130380 Escherichia coli

An additional enzyme that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase which transforms malonyl-CoA tomalonic semialdehyde. Although the aldehyde dehydrogenase functionalityof these enzymes is similar to the bifunctional dehydrogenase fromChlorojlexus aurantiacus, there is little sequence similarity. Bothmalonyl-CoA reductase enzyme candidates have high sequence similarity toaspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reductionand concurrent dephosphorylation of aspartyl-4-phosphate to aspartatesemialdehyde. Additional gene candidates can be found by sequencehomology to proteins in other organisms including Sulfolobussolfataricus and Sulfolobus acidocaldarius and have been listed below.Yet another candidate for CoA-acylating aldehyde dehydrogenase is theald gene from Clostridium beijerinckii reported to reduce acetyl-CoA andbutyryl-CoA to their corresponding aldehydes.

Protein GenBank ID GI Number Organism Msed_0709 YP_001190808.1 146303492Metallosphaera sedula Mcr NP_378167.1 15922498 Sulfolobus tokodaii asd-2NP_343563.1 15898958 Sulfolobus solfataricus Saci_2370 YP_256941.170608071 Sulfolobus acidocaldarius Ald AAT66436 49473535 Clostridiumbeijerinckii eutE AAA80209 687645 Salmonella typhimurium

4-Hydroxybutyryl-CoA reductase catalyzes the reduction of4-hydroxybutyryl-CoA to its corresponding aldehyde. Several acyl-CoAdehydrogenases are capable of catalyzing this activity. The succinatesemialdehyde dehydrogenases (SucD) of Clostridium kluyveri and P.gingivalis were shown in ref (WO2008/115840) to convert4-hydroxybutyryl-CoA to 4-hydroxybutanal as part of a pathway to produce1,4-butanediol. Many butyraldehyde dehydrogenases are also active on4-hydroxybutyraldehyde, including bld of Clostridiumsaccharoperbutylacetonicum and bphG of Pseudomonas sp (Powlowski et al.,J. Bacterial. 175:377-385 (1993)). These and additional proteins with4-hydroxybutyryl-CoA reductase activity are identified below.

Protein GenBank ID GI Number Organism bphG BAA03892.1 425213 Pseudomonassp ald YP_001310903.1 150018649 Clostridium beijerinckii NCIMB 8052 AldZP_03778292.1 225569267 Clostridium hylemonae DSM 15053 AldZP_03705305.1 225016072 Clostridium methylpentosum DSM 5476 AldZP_03715465.1 225026273 Eubacterium hallii DSM 3353 Ald ZP_01962381.1153809713 Ruminococcus obeum ATCC 29174 Ald YP_003701164.1 297585384Bacillus selenitireducens MLS10 Ald AAP42563.1 31075383 Clostridiumsaccharoperbutylacetonicum N1-4 Ald YP_795711.1 116334184 Lactobacillusbrevis ATCC 367 Ald YP_002434126.1 218782808 Desulfatibacillumalkenivorans AK-01 Ald YP_001558295.1 160879327 Clostridiumphytofermentans ISDg Ald ZP_02089671.1 160942363 Clostridium bolteaeATCC BAA-613 Ald ZP_01222600.1 90414628 Photobacterium profundum 3TCKAld YP_001452373.1 157145054 Citrobacter koseri ATCC BAA-895 AldNP_460996.1 16765381 Salmonella enterica typhimurium Ald YP_003307836.1269119659 Sebaldella termitidis ATCC 33386 Ald ZP_04969437.1 254302079Fusobacterium nucleatum subsp. polymorphum ATCC 10953 Ald YP_002892893.1237808453 Tolumonas auensis DSM 9187 Ald YP_426002.1 83592250Rhodospirillum rubrum ATCC 11170

F. 3-Hydroxybutyryl-CoA Hydrolase, Transferase or Synthetase.

An EC 3.1.2.a CoA hydrolase, EC 2.8.3.a CoA transferase, and/or an EC6.2.1.a CoA synthetase provide suitable enzyme activity. See herein.

G. 3-Hydroxybutyrate Reductase.

An EC 1.2.1.e Oxidoreductase (acid to aldehyde) provides suitableactivity. See herein.

H. 3-Hydroxybutyraldehyde Reductase.

An EC 1.1.1.a Oxidoreductase (oxo to alcohol) provides suitableactivity. See herein.

I. Chemical Dehydration or Alternatively See Corresponding EnzymaticPathway in FIG. 6.

J. 3-Hydroxybutyryl-CoA Dehydratase.

An EC 4.2.1. Hydro-lyase provides suitable enzyme activity, and aredescribed below and herein. The enoyl-CoA hydratase of Pseudomonasputida, encoded by ech, catalyzes the conversion of 3-hydroxybutyryl-CoAto crotonyl-CoA (Roberts et al., Arch. Microbiol 117:99-108 (1978)).This transformation is also catalyzed by the crt gene product ofClostridium acetobutylicum, the crt1 gene product of C. kluyveri, andother clostridial organisms Atsumi et al., Metab Eng 10:305-311 (2008);Boynton et al., J Bacterial. 178:3015-3024 (1996); Hillmer et al., FEBSLett. 21:351-354 (1972)). Additional enoyl-CoA hydratase candidates aredescribed below.

Protein GenBank No. GI No. Organism ech NP_745498.1 26990073 Pseudomonasputida crt NP_349318.1 15895969 Clostridium acetobutylicum crt1YP_001393856 153953091 Clostridium kluyveri phaA ABF82233.1 26990002Pseudomonas putida phaB ABF82234.1 26990001 Pseudomonas putida paaANP_745427.1 106636093 Pseudomonas fluorescens paaB NP_745426.1 106636094Pseudomonas fluorescens maoC NP_415905.1 16129348 Escherichia coli paaFNP_415911.1 16129354 Escherichia coli paaG NP_415912.1 16129355Escherichia coli

K. Crotonyl-CoA Reductase (Aldehyde Forming).

An EC 1.2.1.b Oxidoreductase (acyl-CoA to aldehyde) provides suitableenzyme activity. Acyl-CoA reductases in the 1.2.1 family reduce anacyl-CoA to its corresponding aldehyde. Several acyl-CoA reductaseenzymes have been described in the open literature and representsuitable candidates for this step. These are described above.

L. Crotonyl-CoA Hydrolase, Transferase or Synthetase.

An EC 3.1.2.a CoA hydrolase, EC 2.8.3.a CoA transferase, and/or an EC6.2.1.a CoA synthetase provide suitable enzyme activity, and aredescribed in the following sections.

EC 3.1.2.a CoA Hydrolase.

Enzymes in the 3.1.2 family hydrolyze acyl-CoA molecules to theircorresponding acids. Several such enzymes have been described in theliterature and represent suitable candidates for these steps.

For example, the enzyme encoded by acot12 from Rattus norvegicus brain(Robinson et al., Biochem. Biophys. Res. Commun. 71:959-965 (1976)) canreact with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. Exemplary enzymesare shown below.

Protein GenBank Accession No. GI Number Organism acot12 NP_570103.118543355 Rattus norvegicus tesB NP_414986 16128437 Escherichia coliacot8 CAA15502 3191970 Homo sapiens acot8 NP_570112 51036669 Rattusnorvegicus tesA NP_415027 16128478 Escherichia coli ybgC NP_41526416128711 Escherichia coli paaI NP_415914 16129357 Escherichia coli ybdBNP_415129 16128580 Escherichia coli ACH1 NP_009538 6319456 Saccharomycescerevisiae

Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA hydrolasewhich has been described to efficiently catalyze the conversion of3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate during valinedegradation (Shimomura et al., J Biol Chem. 269:14248-14253 (1994)).Genes encoding this enzyme include those below.

Protein GenBank No. GI Number Organism hibch Q5XIE6.2 146324906 Rattusnorvegicus hibch Q6NVY1.2 146324905 Homo sapiens hibch P28817.2 2506374Saccharomyces cerevisiae BC_2292 AP09256 29895975 Bacillus cereus

EC 2.8.3.a CoA Transferase.

Enzymes in the 2.8.3 family catalyze the reversible transfer of a CoAmoiety from one molecule to another. Several CoA transferase enzymeshave been described in the open literature and represent suitablecandidates for these steps. These are described below.

Many transferases have broad specificity and thus can utilize CoAacceptors as diverse as acetate, succinate, propionate, butyrate,2-methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate,crotonate, 3-mercaptopropionate, propionate, vinylacetate, butyrate,among others.

Protein GenBank ID GI Number Organism Ach1 AAX19660.1 60396828 Roseburiasp. A2-183 ROSINTL182_07121 ZP_04743841.2 257413684 Roseburiaintestinalis L1-82 ROSEINA2194_03642 ZP_03755203.1 225377982 Roseburiainulinivorans EUBREC_3075 YP_002938937.1 238925420 Eubacterium rectaleATCC 33656 Pct CAB77207.1 7242549 Clostridium propionicum NT01CX_2372YP_878445.1 118444712 Clostridium novyi NT Cbei_4543 YP_001311608.1150019354 Clostridium beijerinckii CBC_A0889 ZP_02621218.1 168186583Clostridium botulinum C str. Eklund ygfH NP_417395.1 16130821Escherichia coli CIT292_04485 ZP_03838384.1 227334728 Citrobacteryoungae ATCC 29220 SARI_04582 YP_001573497.1 161506385 Salmonellaenterica subsp. arizonae serovar yinte0001_14430 ZP_04635364.1 238791727Yersinia intermedia ATCC 29909

An additional candidate enzyme are identified below.

Protein GenBank ID GI Number Organism pcaI AAN69545.1 24985644Pseudomonas putida pcaJ NP_746082.1 26990657 Pseudomonas putida pcaIYP_046368.1 50084858 Acinetobacter sp. ADP1 pcaJ AAC37147.1 141776Acinetobacter sp. ADP1 pcaI NP_630776.1 21224997 Streptomyces coelicolorpcaJ NP_630775.1 21224996 Streptomyces coelicolor HPAG1_0676 YP_627417108563101 Helicobacter pylori HPAG1_0677 YP_627418 108563102Helicobacter pylori ScoA NP_391778 16080950 Bacillus subtilis ScoBNP_391777 16080949 Bacillus subtilis

A CoA transferase that can utilize acetate as the CoA acceptor isacetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit)and atoD (beta subunit) genes. Similar enzymes are identified below.

Protein GenBank ID GI Number Organism atoA P76459.1 2492994 Escherichiacoli K12 atoD P76458.1 2492990 Escherichia coli K12 actA YP_226809.162391407 Corynebacterium glutamicum ATCC 13032 cg0592 YP_224801.162389399 Corynebacterium glutamicum ATCC 13032 ctfA NP_149326.1 15004866Clostridium acetobutylicum ctfB NP_149327.1 15004867 Clostridiumacetobutylicum ctfA AAP42564.1 31075384 Clostridiumsaccharoperbutylacetonicum ctfB AAP42565.1 31075385 Clostridiumsaccharoperbutylacetonicum

Additional exemplary transferase candidates are catalyzed by the geneproducts of cat1, cat2, and cat3 of Clostridium kluyveri which have beenshown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoAtransferase activity, respectively. Similar CoA transferase activitiesare identified below.

Protein GenBank ID GI Number Organism cat1 P38946.1 729048 Clostridiumkluyveri cat2 P38942.2 172046066 Clostridium kluyveri cat3 EDK35586.1146349050 Clostridium kluyveri TVAG_395550 XP_001330176 123975034Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875 Trypanosomabrucei

The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobicbacterium Acidaminococcus fermentans reacts with diacid glutaconyl-CoAand 3-butenoyl-CoA (Mack et al., FEBS Lett. 405:209-212 (1997)). Thegenes encoding this enzyme are gctA and gctB. These proteins areidentified below.

Protein GenBank ID GI Number Organism gctA CAA57199.1 559392Acidaminococcus fermentans gctB CAA57200.1 559393 Acidaminococcusfermentans

EC 6.2.1.a CoA Synthase (Acid-Thiol Ligase).

The conversion of acyl-CoA substrates to their acid products can becatalyzed by a CoA acid-thiol ligase or CoA synthetase in the 6.2.1family of enzymes, several of which are reversible. Several enzymescatalyzing CoA acid-thiol ligase or CoA synthetase activities have beendescribed in the literature and represent suitable candidates for thesesteps.

For example, ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is anenzyme that couples the conversion of acyl-CoA esters to theircorresponding acids with the concomitant synthesis of ATP. The enzymesfrom A. fulgidus, H marismortui and P. aerophilum have all been cloned,functionally expressed, and characterized in E. coli (Brasen andSchonheit, supra; Musfeldt and Schonheit, J Bacteriol. 184:636-644(2002)). An additional candidate is succinyl-CoA synthetase, encoded bysucCD of E. coli and LSC1 and LSC2 genes of Saccharomyces cerevisiae.These enzymes catalyze the formation of succinyl-CoA from succinate withthe concomitant consumption of one ATP in a reaction which is reversiblein vivo (Buck et al., Biochemistry 24:6245-6252 (1985)). Exemplaryenzyme are below.

Protein GenBank ID GI Number Organism AF1211 NP_070039.1 11498810Archaeoglobus fulgidus AF1983 NP_070807.1 11499565 Archaeoglobusfulgidus Scs YP_135572.1 55377722 Haloarcula marismortui PAE3250NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2 sucC NP_415256.116128703 Escherichia coli sucD AAC73823.1 1786949 Escherichia coli LSC1NP_014785 6324716 Saccharomyces cerevisiae LSC2 NP_011760 6321683Saccharomyces cerevisiae paaF AAC24333.2 22711873 Pseudomonas putidamatB AAC83455.1 3982573 Rhizobium leguminosarum

Another candidate enzyme for these steps is 6-carboxyhexanoate-CoAligase, also known as pimeloyl-CoA ligase (EC 6.2.1.14), which naturallyactivates pimelate to pimeloyl-CoA during biotin biosynthesis ingram-positive bacteria. Exemplary enzymes are below.

Protein GenBank ID GI Number Organism bioW NP_390902.2 50812281 Bacillussubtilis bioW CAA10043.1 3850837 Pseudomonas mendocina bioW P22822.1115012 Bacillus sphaericus

Additional CoA-ligases are listed below.

Protein Accession No. GI No. Organism phl CAJ15517.1 77019264Penicillium chrysogenum phlB ABS19624.1 152002983 Penicilliumchrysogenum paaF AAC24333.2 22711873 Pseudomonas putida bioW NP_390902.250812281 Bacillus subtilis AACS NP_084486.1 21313520 Mus musculus AACSNP_076417.2 31982927 Homo sapiens

Like enzymes in other classes, certain enzymes in the EC class 6.2.1have been determined to have broad substrate specificity. The acyl CoAligase from Pseudomonas putida has been demonstrated to work on severalaliphatic substrates including acetic, propionic, butyric, valeric,hexanoic, heptanoic, and octanoic acids and on aromatic compounds suchas phenylacetic and phenoxyacetic acids (Femandez-Valverde et al.,Applied and Environmental Microbiology 59:1149-1154 (1993)). A relatedenzyme, malonyl CoA synthetase (6.3.4.9) from Rhizobium trifoli couldconvert several diacids, namely, ethyl-, propyl-, allyl-, isopropyl-,dimethyl-, cyclopropyl-, cyclopropylmethylene-, cyclobutyl-, andbenzyl-malonate into their corresponding monothioesters (Pohl et al., J.Am. Chem. Soc. 123:5822-5823 (2001)).

M. Crotonate Reductase.

A suitable enzyme activity is an 1.2.1.e Oxidoreductase (acid toaldehyde), which include the following.

The conversion of an acid to an aldehyde is thermodynamicallyunfavorable and typically requires energy-rich cofactors and multipleenzymatic steps. Direct conversion of the acid to aldehyde by a singleenzyme is catalyzed by an acid reductase enzyme in the 1.2.1 family.Exemplary acid reductase enzymes include carboxylic acid reductase,alpha-aminoadipate reductase and retinoic acid reductase. Carboxylicacid reductase (CAR), found in Nocardia iowensis, catalyzes themagnesium, ATP and NADPH-dependent reduction of carboxylic acids totheir corresponding aldehydes. CAR requires post-translationalactivation by a phosphopantetheine transferase (PPTase) that convertsthe inactive apo-enzyme to the active holo-enzyme (Hansen et al., Appl.Environ. Microbiol 75:2765-2774 (2009)). Expression of the npt gene,encoding a specific PPTase, product improved activity of the enzyme.Exemplary enzymes include those below.

Gene GenBank Accession No. GI No. Organism car AAR91681.1 40796035Nocardia iowensis npt ABI83656.1 114848891 Nocardia iowensis griCYP_001825755.1 182438036 Streptomyces griseus griD YP_001825756.1182438037 Streptomyces griseus

Additional car and npt genes can be identified based on sequencehomology.

Gene name GI No. GenBank Accession No. Organism fadD9 121638475YP_978699.1 Mycobacterium bovis BCG BCG_2812c 121638674 YP_978898.1Mycobacterium bovis BCG nfa20150 54023983 YP_118225.1 Nocardia farcinicaIFM 10152 nfa40540 54026024 YP_120266.1 Nocardia farcinica IFM 10152SGR_6790 182440583 YP_001828302.1 Streptomyces griseus subsp. griseusNBRC 13350 SGR_665 182434458 YP_001822177.1 Streptomyces griseus subsp.griseus NBRC 13350 MSMEG_2956 YP_887275.1 YP_887275.1 Mycobacteriumsmegmatis MC2 155 MSMEG_5739 YP_889972.1 118469671 Mycobacteriumsmegmatis MC2 155 MSMEG_2648 YP_886985.1 118471293 Mycobacteriumsmegmatis MC2 155 MAP1040C NP_959974.1 41407138 Mycobacterium aviumsubsp. paratuberculosis K-10 MAP2899C NP_961833.1 41408997 Mycobacteriumavium subsp. paratuberculosis K-10 MMAR_2117 YP_001850422.1 183982131Mycobacterium marinum M MMAR_2936 YP_001851230.1 183982939 Mycobacteriummarinum M MMAR_1916 YP_001850220.1 183981929 Mycobacterium marinum MTpauDRAFT_33060 ZP_04027864.1 227980601 Tsukamurella paurometabola DSM20162 TpauDRAFT_20920 ZP_04026660.1 ZP_04026660.1 Tsukamurellapaurometabola DSM 20162 CPCC7001_1320 ZP_05045132.1 254431429 CyanobiumPCC7001 DDBDRAFT_0187729 XP_636931.1 66806417 Dictyostelium discoideumAX4

An enzyme with similar characteristics, alpha-aminoadipate reductase(AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in somefungal species. Like CAR, this enzyme utilizes magnesium and requiresactivation by a PPTase. Enzyme candidates for AAR and its correspondingPPTase are below.

Gene GenBank Accession No. GI No. Organism LYS2 AAA34747.1 171867Saccharomyces cerevisiae LYS5 P50113.1 1708896 Saccharomyces cerevisiaeLYS2 AAC02241.1 2853226 Candida albicans LYS5 AAO26020.1 28136195Candida albicans Lys1p P40976.3 13124791 Schizosaccharomyces pombe Lys7pQ10474.1 1723561 Schizosaccharomyces pombe Lys2 CAA74300.1 3282044Penicillium chrysogenum

N. Crotonaldehyde Reductase.

A suitable enzyme activity is provided by an EC 1.1.1.a Oxidoreductase(oxo to alcohol). EC 1.1.1.a Oxidoreductase (oxo to alcohol) includesthe following:

The reduction of glutarate semialdehyde to 5-hydroxyvalerate byglutarate semialdehyde reductase entails reduction of an aldehyde to itscorresponding alcohol. Enzymes with glutarate semialdehyde reductaseactivity include those below.

PROTEIN GENBANK ID GI NUMBER ORGANISM ATEG_00539 XP_001210625.1115491995 Aspergillus terreus NIH2624 4hbd AAK94781.1 15375068Arabidopsis thaliana

Additional genes encoding enzymes that catalyze the reduction of analdehyde to alcohol (i.e., alcohol dehydrogenase or equivalentlyaldehyde reductase) include those below. The enzyme candidates describedpreviously for catalyzing the reduction of methylglyoxal to acetol orlactaldehyde are also suitable lactaldehyde reductase enzyme candidates.

Protein GENBANK ID GI NUMBER ORGANISM alrA BAB12273.1 9967138Acinetobacter sp. strain M-1 ADH2 NP_014032.1 6323961 Saccharomycescerevisiae yqhD NP_417484.1 16130909 Escherichia coli fucO NP_417279.116130706 Escherichia coli bdh I NP_349892.1 15896543 Clostridiumacetobutylicum bdh II NP_349891.1 15896542 Clostridium acetobutylicumadhA YP_162971.1 56552132 Zymomonas mobilis bdh BAF45463.1 124221917Clostridium saccharoperbutylacetonicum Cbei_1722 YP_001308850 150016596Clostridium beijerinckii Cbei_2181 YP_001309304 150017050 Clostridiumbeijerinckii Cbei_2421 YP_001309535 150017281 Clostridium beijerinckiiGRE3 P38715.1 731691 Saccharomyces cerevisiae ALD2 CAA89806.1 825575Saccharomyces cerevisiae ALD3 NP_013892.1 6323821 Saccharomycescerevisiae ALD4 NP_015019.1 6324950 Saccharomyces cerevisiae ALD5NP_010996.2 330443526 Saccharomyces cerevisiae ALD6 ABX39192.1 160415767Saccharomyces cerevisiae HFD1 Q04458.1 2494079 Saccharomyces cerevisiaeGOR1 NP_014125.1 6324055 Saccharomyces cerevisiae YPL113C AAB68248.11163100 Saccharomyces cerevisiae GCY1 CAA99318.1 1420317 Saccharomycescerevisiae

Enzymes exhibiting 4-hydroxybutymte dehydrogenase activity (EC 1.1.1.61)also fall into this category and exemplary enzymes are below.

PROTEIN GENBANK ID GI NUMBER ORGANISM 4hbd YP_726053.1 113867564Ralstonia eutropha H16 4hbd L21902.1 146348486 Clostridium kluyveri DSM555 adhI AAR91477.1 40795502 Geobacillus thermoglucosidasius

Another exemplary aldehyde reductase is methylmalonate semialdehydereductase, also known as 3-hydroxyisobutyrate dehydrogenase (EC1.1.1.31). This enzyme participates in valine, leucine and isoleucinedegradation and has been identified in bacteria, eukaryotes, andmammals. Exemplary enzymes include those below.

PROTEIN GENBANK ID GI NUMBER ORGANISM P84067 P84067 75345323 Thermusthermophilus 3hidh P31937.2 12643395 Homo sapiens 3hidh P32185.1 416872Oryctolagus cuniculus mmsB NP_746775.1 26991350 Pseudomonas putida mmsBP28811.1 127211 Pseudomonas aeruginosa dhat Q59477.1 2842618 Pseudomonasputida

There exist several exemplary alcohol dehydrogenases that convert aketone to a hydroxyl functional group. Two such enzymes from E. coli areencoded by malate dehydrogenase (mdh) and lactate dehydrogenase (ldhA).Exemplary enzymes include those below.

Protein Genbank ID GI Number Organism mdh AAC76268.1 1789632 Escherichiacoli ldhA NP_415898.1 16129341 Escherichia coli ldh YP_725182.1113866693 Ralstonia eutropha bdh AAA58352.1 177198 Homo sapiens adhAAA23199.2 60592974 Clostridium beijerinckii NRRL B593 adh P14941.1113443 Thermoanaerobacter brockii HTD4 sadh CAD36475 21615553Rhodococcus ruber adhA AAC25556 3288810 Pyrococcus furiosus

A number of organisms encode genes that catalyze the reduction of3-oxobutanol to 1,3-butanediol, including those belonging to the genusBacillus, Brevibacterium, Candida, and Klebsiella among others, asdescribed by Matsuyama et al. J Mol Cat B Enz, 11:513-521 (2001). One ofthese enzymes, SADH from Candida parapsilosis, was cloned andcharacterized in E. coli. A mutated Rhodococcus phenylacetaldehydereductase (Sar268) and a Leifonia alcohol dehydrogenase have also beenshown to catalyze this transformation at high yields (Itoh et al.,Appalicrobiol Biotechnol. 75:1249-1256 (2007)).

Protein Genbank ID GI Number Organism sadh BAA24528.1 2815409 Candidaparapsilosis

O. Crotyl Alcohol Kinase.

Crotyl alcohol kinase enzymes catalyze the transfer of a phosphate groupto the hydroxyl group of crotyl alcohol. The enzymes described belownaturally possess such activity or can be engineered to exhibit thisactivity. Kinases that catalyze transfer of a phosphate group to analcohol group are members of the EC 2.7.1 enzyme class. The table belowlists several useful kinase enzymes in the EC 2.7.1 enzyme class.

Enzyme Commission Number Enzyme Name 2.7.1.1 hexokinase 2.7.1.2glucokinase 2.7.1.3 ketohexokinase 2.7.1.4 fructokinase 2.7.1.5rhamnulokinase 2.7.1.6 galactokinase 2.7.1.7 mannokinase 2.7.1.8glucosamine kinase 2.7.1.10 phosphoglucokinase 2.7.1.116-phosphofructokinase 2.7.1.12 gluconokinase 2.7.1.13dehydrogluconokinase 2.7.1.14 sedoheptulokinase 2.7.1.15 ribokinase2.7.1.16 ribulokinase 2.7.1.17 xylulokinase 2.7.1.18 phosphoribokinase2.7.1.19 phosphoribulokinase 2.7.1.20 adenosine kinase 2.7.1.21thymidine kinase 2.7.1.22 ribosylnicotinamide kinase 2.7.1.23 NAD+kinase 2.7.1.24 dephospho-CoA kinase 2.7.1.25 adenylyl-sulfate kinase2.7.1.26 riboflavin kinase 2.7.1.27 erythritol kinase 2.7.1.28triokinase 2.7.1.29 glycerone kinase 2.7.1.30 glycerol kinase 2.7.1.31glycerate kinase 2.7.1.32 choline kinase 2.7.1.33 pantothenate kinase2.7.1.34 pantetheine kinase 2.7.1.35 pyridoxal kinase 2.7.1.36mevalonate kinase 2.7.1.39 homoserine kinase 2.7.1.40 pyruvate kinase2.7.1.41 glucose-1-phosphate phosphodismutase 2.7.1.42 riboflavinphosphotransferase 2.7.1.43 glucuronokinase 2.7.1.44 galacturonokinase2.7.1.45 2-dehydro-3-deoxygluconokinase 2.7.1.46 L-arabinokinase2.7.1.47 D-ribulokinase 2.7.1.48 uridine kinase 2.7.1.49hydroxymethylpyrimidine kinase 2.7.1.50 hydroxyethylthiazole kinase2.7.1.51 L-fuculokinase 2.7.1.52 fucokinase 2.7.1.53 L-xylulokinase2.7.1.54 D-arabinokinase 2.7.1.55 allose kinase 2.7.1.561-phosphofructokinase 2.7.1.58 2-dehydro-3-deoxygalactonokinase 2.7.1.59N-acetylglucosamine kinase 2.7.1.60 N-acylmannosamine kinase 2.7.1.61acyl-phosphate-hexose phosphotransferase 2.7.1.62 phosphoramidate-hexosephosphotransferase 2.7.1.63 polyphosphate-glucose phosphotransferase2.7.1.64 inositol 3-kinase 2.7.1.65 scyllo-inosamine 4-kinase 2.7.1.66undecaprenol kinase 2.7.1.67 1-phosphatidylinositol 4-kinase 2.7.1.681-phosphatidylinositol-4-phosphate 5-kinase 2.7.1.69protein-Np-phosphohistidine-sugar phosphotransferase 2.7.1.70 identicalto EC 2.7.1.37. 2.7.1.71 shikimate kinase 2.7.1.72 streptomycin 6-kinase2.7.1.73 inosine kinase 2.7.1.74 deoxycytidine kinase 2.7.1.76deoxyadenosine kinase 2.7.1.77 nucleoside phosphotransferase 2.7.1.78polynucleotide 5′-hydroxyl-kinase 2.7.1.79 diphosphate-glycerolphosphotransferase 2.7.1.80 diphosphate-serine phosphotransferase2.7.1.81 hydroxylysine kinase 2.7.1.82 ethanolamine kinase 2.7.1.83pseudouridine kinase 2.7.1.84 alkylglycerone kinase 2.7.1.85 β-glucosidekinase 2.7.1.86 NADH kinase 2.7.1.87 streptomycin 3″-kinase 2.7.1.88dihydrostreptomycin-6-phosphate 3′a-kinase 2.7.1.89 thiamine kinase2.7.1.90 diphosphate-fructose-6-phosphate 1-phosphotransferase 2.7.1.91sphinganine kinase 2.7.1.92 5-dehydro-2-deoxygluconokinase 2.7.1.93alkylglycerol kinase 2.7.1.94 acylglycerol kinase 2.7.1.95 kanamycinkinase 2.7.1.100 S-methyl-5-thioribose kinase 2.7.1.101 tagatose kinase2.7.1.102 hamamelose kinase 2.7.1.103 viomycin kinase 2.7.1.1056-phosphofructo-2-kinase 2.7.1.106 glucose-1,6-bisphosphate synthase2.7.1.107 diacylglycerol kinase 2.7.1.108 dolichol kinase 2.7.1.113deoxyguanosine kinase 2.7.1.114 AMP-thymidine kinase 2.7.1.118ADP-thymidine kinase 2.7.1.119 hygromycin-B 7″-O-kinase 2.7.1.121phosphoenolpyruvate-glycerone phosphotransferase 2.7.1.122 xylitolkinase 2.7.1.127 inositol-trisphosphate 3-kinase 2.7.1.130tetraacyldisaccharide 4′-kinase 2.7.1.134 inositol-tetrakisphosphate1-kinase 2.7.1.136 macrolide 2′-kinase 2.7.1.137 phosphatidylinositol3-kinase 2.7.1.138 ceramide kinase 2.7.1.140 inositol-tetrakisphosphate5-kinase 2.7.1.142 glycerol-3-phosphate-glucosephosphotransferase2.7.1.143 diphosphate-purine nucleoside kinase 2.7.1.144tagatose-6-phosphate kinase 2.7.1.145 deoxynucleoside kinase 2.7.1.146ADP-dependent phosphofructokinase 2.7.1.147 ADP-dependent glucokinase2.7.1.148 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase2.7.1.149 1-phosphatidylinositol-5-phosphate 4-kinase 2.7.1.1501-phosphatidylinositol-3-phosphate 5-kinase 2.7.1.151inositol-polyphosphate multikinase 2.7.1.153phosphatidylinositol-4,5-bisphosphate 3-kinase 2.7.1.154phosphatidylinositol-4-phosphate 3-kinase 2.7.1.156 adenosylcobinamidekinase 2.7.1.157 N-acetylgalactosamine kinase 2.7.1.158inositol-pentakisphosphate 2-kinase 2.7.1.159inositol-1,3,4-trisphosphate 5/6-kinase 2.7.1.160 2′-phosphotransferase2.7.1.161 CTP-dependent riboflavin kinase 2.7.1.162 N-acetylhexosamine1-kinase 2.7.1.163 hygromycin B 4-O-kinase 2.7.1.164O-phosphoseryl-tRNASec kinase

Mevalonate kinase (EC 2.7.1.36) phosphorylates the terminal hydroxylgroup of mevalonate. Gene candidates for this step include erg12 from S.cerevisiae, mvk from Methanocaldococcus jannaschi, MVK from Homosapiens, and mvk from Arabidopsis thaliana col. Additional mevalonatekinase candidates include the feedback-resistant mevalonate kinase fromthe archeon Methanosarcina mazei (Primak et al, AEM, in press (2011))and the Mvk protein from Streptococcus pneumoniae (Andreassi et al,Protein Sci, 16:983-9 (2007)). Exemplary Mvk proteins are below.

Protein GenBank ID GI Number Organism erg12 CAA39359.1 3684Sachharomyces cerevisiae mvk Q58487.1 2497517 Methanocaldococcusjannaschii mvk AAH16140.1 16359371 Homo sapiens mvk NP_851084.1 30690651Arabidopsis thaliana mvk NP_633786.1 21227864 Methanosarcina mazei mvkNP_357932.1 15902382 Streptococcus pneumoniae

Glycerol kinase also phosphorylates the terminal hydroxyl group inglycerol to form glycerol-3-phosphate. This reaction occurs in severalspecies, including Escherichia coli, Saccharomyces cerevisiae, andThermotoga maritima. Glycerol kinases have been shown to have a widerange of substrate specificity. Crans and Whiteside studied glycerolkinases from four different organisms (Escherichia coli, S. cerevisiae,Bacillus stearothermophilus, and Candida mycoderma) (Crans et al., J.Am. Chem. Soc. 107:7008-7018 (2010); Nelson et al., supra, (1999)). Theystudied 66 different analogs of glycerol and concluded that the enzymecould accept a range of substituents in place of one terminal hydroxylgroup and that the hydrogen atom at C2 could be replaced by a methylgroup. Interestingly, the kinetic constants of the enzyme from all fourorganisms were very similar.

Protein GenBank ID GI Number Organism glpK AP_003883.1 89110103Escherichia coli K12 glpK1 NP_228760.1 15642775 Thermotoga maritime MSB8glpK2 NP_229230.1 15642775 Thermotoga maritime MSB8 Gut1 NP_011831.182795252 Saccharomyces cerevisiae

Homoserine kinase is another possible candidate. This enzyme is alsopresent in a number of organisms including E. coli, Streptomyces sp, andS. cerevisiae. The gene candidates are:

Protein GenBank ID GI Number Organism thrB BAB96580.2 85674277Escherichia coli K12 SACT1DRAFT_4809 ZP_06280784.1 282871792Streptomyces sp. ACT-1 Thr1 AAA35154.1 172978 Saccharomyces serevisiae

P. 2-Butenyl-4-Phosphate Kinase.

2-Butenyl-4-phosphate kinase enzymes catalyze the transfer of aphosphate group to the phosphate group of 2-butenyl-4-phosphate. Theenzymes described below naturally possess such activity or can beengineered to exhibit this activity. Kinases that catalyze transfer of aphosphate group to another phosphate group are members of the EC 2.7.4enzyme class. The table below lists several useful kinase enzymes in theEC 2.7.4 enzyme class.

Enzyme Commission Number Enzyme Name 2.7.4.1 polyphosphate kinase2.7.4.2 phosphomevalonate kinase 2.7.43 adenylate kinase 2.7.4.4nucleoside-phosphate kinase 2.7.4.6 nucleoside-diphosphate kinase2.7.4.7 phosphomethylpyrimidine kinase 2.7.4.8 guanylate kinase 2.7.4.9dTMP kinase 2.7.4.10 nucleoside-triphosphate-adenylate kinase 2.7.4.11(deoxy)adenylate kinase 2.7.4.12 T2-induced deoxynucleotide kinase2.7.4.13 (deoxy)nucleoside-phosphate kinase 2.7.4.14 cytidylate kinase2.7.4.15 thiamine-diphosphate kinase 2.7.4.16 thiamine-phosphate kinase2.7.4.17 3-phosphoglyceroyl-phosphate- polyphosphate phosphotransferase2.7.4.18 farnesyl-diphosphate kinase 2.7.4.19 5-methyldeoxycytidine-5′-phosphate kinase 2.7.4.20 dolichyl-diphosphate-polyphosphatephosphotransferase 2.7.4.21 inositol-hexakisphosphate kinase 2.7.4.22UMP kinase 2.7.4.23 ribose 1,5-bisphosphate phosphokinase 2.7.4.24diphosphoinositol-pentakisphosphate kinase 2.7.4.— Farnesylmonophosphate kinase 2.7.4.— Geranyl-geranyl monophosphate kinase2.7.4.— Phytyl-phosphate kinase

Phosphomevalonate kinase enzymes are of particular interest.Phosphomevalonate kinase (EC 2.7.4.2) catalyzes the analogoustransformation to 2-butenyl-4-phosphate kinase. Exemplary enzymesinclude those below.

Protein GenBank ID GI Number Organism Erg8 AAA34596.1 171479Saccharomyces cerevisiae mvaK2 AAG02426.1 9937366 Staphylococcus aureusmvaK2 AAG02457.1 9937409 Streptococcus pneumoniae mvaK2 AAG02442.19937388 Enterococcus faecalis

Famesyl monophosphate kinase enzymes catalyze the CTP dependentphosphorylation of famesyl monophosphate to famesyl diphosphate.Similarly, geranylgeranyl phosphate kinase catalyzes CTP dependentphosphorylation. Enzymes with these activities were identified in themicrosomal fraction of cultured Nicotiana tabacum (Thai et al, PNAS96:13080-5 (1999)). However, the associated genes have not beenidentified to date.

Q. Butadiene Synthase.

Butadiene synthase catalyzes the conversion of 2-butenyl-4-diphosphateto 1,3-butadiene. The enzymes described below naturally possess suchactivity or can be engineered to exhibit this activity. Carbon-oxygenlyases that operate on phosphates are found in the EC 4.2.3 enzymeclass. The table below lists several useful enzymes in EC class 4.2.3.

Enzyme Commission Number Enzyme Name 4.2.3.15 Myrcene synthase 4.2.3.26Linalool synthase 4.2.3.27 Isoprene synthase 4.2.3.36 Terpentrienesythase 4.2.3.46 (E, E)-alpha-Farnesene synthase 4.2.3.47 Beta-Farnesenesynthase 4.2.3.49 Nerolidol synthase

Particularly useful enzymes include isoprene synthase, myrcene synthaseand famesene synthase. Enzyme candidates are described below.

Isoprene synthase naturally catalyzes the conversion of dimethylallyldiphosphate to isoprene, but can also catalyze the synthesis of1,3-butadiene from 2-butenyl-4-diphosphate. Isoprene synthases can befound in several organisms including Populus alba (Sasaki et al., FEBSLetters, 2005, 579 (11), 2514-2518), Pueraria montana (Lindberg et al.,Metabolic Eng, 12(1):70-79 (2010); Sharkey et al., Plant Physiol.,137(2):700-712 (2005)), and Populus tremula x Populus alba, also calledPopulus canescens (Miller et al., Planta, 2001, 213 (3), 483-487). Thecrystal structure of the Populus canescens isoprene synthase wasdetermined (Koksal et al, J Mol Biol 402:363-373 (2010)). Additionalisoprene synthase enzymes are described in (Chotani et al.,WO/2010/031079, Systems Using Cell Culture for Production of Isoprene;Cervin et al., US Patent Application 20100003716, Isoprene SynthaseVariants for Improved Microbial Production of Isoprene).

Protein GenBank ID GI Number Organism ispS BAD98243.1 63108310 Populusalba ispS AAQ84170.1 35187004 Pueraria montana ispS CAC35696.1 13539551Populus tremula × Populus alba

Myrcene synthase enzymes catalyze the dephosphorylation of geranyldiphosphate to beta-myrcene (EC 4.2.3.15). Exemplary myrcene synthasesare below. These enzymes were heterologously expressed in E. coli.

Protein GenBank ID GI Number Organism MST2 ACN58229.1 224579303 Solanumlycopersicum TPS-Myr AAS47690.2 77546864 Picea abies G-myr O24474.117367921 Abies grandis TPS10 EC07543.1 330252449 Arabidopsis thaliana

Famesyl diphosphate is converted to alpha-famesene and beta-famesene byalpha-famesene synthase and beta-famesene synthase, respectively.Exemplary alpha-famesene synthase enzymes include those below.

Protein GenBank ID GI Number Organism TPS03 A4FVP2.1 205829248Arabidopsis thaliana TPS02 P0CJ43.1 317411866 Arabidopsis thalianaTPS-Far AAS47697.1 44804601 Picea abies afs AAU05951.1 51537953 Cucumissativus eafar Q84LB2.2 75241161 Malus × domestica TPS1 Q84ZW8.1 75149279Zea mays

R. Crotyl Alcohol Diphosphokinase.

Crotyl alcohol diphosphokinase enzymes catalyze the transfer of adiphosphate group to the hydroxyl group of crotyl alcohol. The enzymesdescribed below naturally possess such activity or can be engineered toexhibit this activity. Kinases that catalyze transfer of a diphosphategroup are members of the EC 2.7.6 enzyme class. The table below listsseveral useful kinase enzymes in the EC 2.7.6 enzyme class.

Enzyme Commission Number Enzyme Name 2.7.6.1 ribose-phosphatediphosphokinase 2.7.6.2 thiamine diphosphokinase 2.7.6.32-amino-4-hydroxy-6- hydroxymethyldihydropteridine diphosphokinase2.7.6.4 nucleotide diphosphokinase 2.7.6.5 GTP diphosphokinase

Of particular interest are ribose-phosphate diphosphokinase enzymes;exemplary enzymes are below.

Protein GenBank ID GI Number Organism prs NP_415725.1 16129170Escherichia coli prsA NP_109761.1 13507812 Mycoplasma pneumoniae M129TPK1 BAH19964.1 222424006 Arabidopsis thaliana col TPK2 BAH57065.1227204427 Arabidopsis thaliana col

S. Chemical Dehydration or Crotyl Alcohol Dehydratase.

Converting crotyl alcohol to butadiene using a crotyl alcoholdehydratase can include combining the activities of the enzymaticisomerization of crotyl alcohol to 3-buten-2-ol then dehydration of3-buten-2-ol to butadiene. An exemplary bifunctional enzyme withisomerase and dehydratase activities is the linalooldehydratase/isomemse of Castellaniella defragrans. This enzyme catalyzesthe isomerization of geraniol to linalool and the dehydration oflinalool to myrcene, reactants similar in structure to crotyl alcohol,3-buten-2-ol and butadiene (Brodkorb et al, J Biol Chem 285:30436-42(2010)). Enzyme accession numbers and homologs are listed in the tablebelow.

Protein GenBank ID GI Number Organism Ldi E1XUJ2.1 403399445Castelleniella defragrans STEHIDRAFT_68678 EIM80109.1 389738914 Stereumhirsutum FP-91666 SS1 NECHADRAFT_82460 XP_003040778.1 302883759 Nectriahaematococca mpVI 77-13-4 AS9A_2751 YP_004493998.1 333920417Amycolicicoccus subflavus DQS3-9A1

Alternatively, a fusion protein or protein conjugate can be generatedusing well know methods in the art to generate a bi-functional(dual-functional) enzyme having both the isomemse and dehydrataseactivities. The fusion protein or protein conjugate can include at leastthe active domains of the enzymes (or respective genes) of the isomemseand dehydratase reactions. For the first step, the conversion of crotylalcohol to 3-buten-2-ol, enzymatic conversion can be catalyzed by acrotyl alcohol isomemse (classified as EC 5.4.4). A similarisomerization, the conversion of 2-methyl-3-buten-2-ol to3-methyl-2-buten-1-ol, is catalyzed by cell extracts of Pseudomonasputida MB-1 (Malone et al, AEM 65 (6): 2622-30 (1999)). The extract maybe used in vitro, or the protein or gene(s) associated with the isomemseactivity can be isolated and used, even though they have not beenidentified to date.

Alternatively, either or both steps can be done by chemical conversion,or by enzymatic conversion (in vivo or in vitro), or any combination.Enzymes having the desired activity for the conversion of 3-buten-2-olto butadiene are provided elsewhere herein.

T. Butadiene Synthase (Monophosphate).

Butadiene synthase (monophosphate) catalyzes the conversion of2-butenyl-4-phosphate to 1,3-butadiene. Butadiene synthase enzymes areof the EC 4.2.3 enzyme class as described herein that possess suchactivity or can be engineered to exhibit this activity. Diphosphatelyase enzymes catalyze the conversion of alkyl diphosphates to alkenes.Carbon-oxygen lyases that operate on phosphates are found in the EC4.2.3 enzyme class. The table below lists several useful enzymes in ECclass 4.2.3. Exemplary enzyme candidates are also phosphate lyases.

Enzyme Commission No. Enzyme Name 4.2.3.5 Chorismate synthase 4.2.3.15Myrcene synthase 4.2.3.27 Isoprene synthase 4.2.3.36 Terpentrienesythase 4.2.3.46 (E, E)-alpha-Farnesene synthase 4.2.3.47 Beta-Farnesenesynthase

Phosphate lyase enzymes catalyze the conversion of alkyl phosphates toalkenes. Carbon-oxygen lyases that operate on phosphates are found inthe EC 4.2.3 enzyme class. The table below lists several relevantenzymes in EC class 4.2.3.

Enzyme Commission Number Enzyme Name 4.2.3.5 Chorismate synthase4.2.3.15 Myrcene synthase 4.2.3.26 Linalool synthase 4.2.3.27 Isoprenesynthase 4.2.3.36 Terpentriene sythase 4.2.3.46 (E, E)-alpha-Farnesenesynthase 4.2.3.47 Beta-Farnesene synthase 4.2.3.49 Nerolidol synthase4.2.3.— Methylbutenol synthase

Isoprene synthase enzymes catalyzes the conversion of dimethylallyldiphosphate to isoprene. Additional isoprene synthase enzymes are belowand are described in (Chotani et al., WO/2010/031079, Systems Using CellCulture for Production of Isoprene; Cervin et al., US Patent Application20100003716, Isoprene Synthase Variants for Improved MicrobialProduction of Isoprene).

Protein GenBank ID GI Number Organism ispS BAD98243.1 63108310 Populusalba ispS AAQ84170.1 35187004 Pueraria montana ispS CAC35696.1 13539551Populus tremula × Populus alba Tps-MBO1 AEB53064.1 328834891 Pinussabiniana

Chorismate synthase (EC 4.2.3.5) participates in the shikimate pathway,catalyzing the dephosphorylation of 5-enolpyruvylshikimate-3-phosphateto chorismate. The enzyme requires reduced flavin mononucleotide (FMN)as a cofactor, although the net reaction of the enzyme does not involvea redox change. In contrast to the enzyme found in plants and bacteria,the chorismate synthase in fungi is also able to reduce FMN at theexpense of NADPH (Macheroux et al., Planta 207:325-334 (1999)).Representative monofunctional enzymes and bifunctional fungal enzymesare below.

Gene GenBank Accession No. GI No. Organism aroC NP_416832.1 16130264Escherichia coli aroC ACH47980.1 197205483 Streptococcus pneumoniaeU25818.1: 19 . . . 1317 AAC49056.1 976375 Neurospora crassa ARO2CAA42745.1 3387 Saccharomyces cerevisiae

Myrcene synthase enzymes catalyze the dephosphorylation of geranyldiphosphate to beta-myrcene (EC 4.2.3.15). Exemplary myrcene synthasesare below. These enzymes were heterologously expressed in E coli.

Protein GenBank ID GI Number Organism MST2 ACN58229.1 224579303 Solanumlycopersicum TPS-Myr AAS47690.2 77546864 Picea abies G-myr O24474.117367921 Abies grandis TPS10 EC07543.1 330252449 Arabidopsis thaliana

Famesyl diphosphate is converted to alpha-famesene and beta-famesene byalpha-famesene synthase and beta-famesene synthase, respectively.Exemplary alpha-famesene synthase enzymes are below.

Protein GenBank ID GI Number Organism TPS03 A4FVP2.1 205829248Arabidopsis thaliana TPS02 P0CJ43.1 317411866 Arabidopsis thalianaTPS-Far AAS47697.1 44804601 Picea abies afs AAU05951.1 51537953 Cucumissativus eafar Q84LB2.2 75241161 Malus × domestica TPS1 Q84ZW8.1 75149279Zea mays

U. Crotonyl-CoA Reductase (Alcohol Forming) and V) 3-Hydroxybutyryl-CoAReductase (Alcohol Forming).

The direct conversion of crotonyl-CoA and 3-hydroxybutyryl-CoAsubstrates to their corresponding alcohols is catalyzed by bifunctionalenzymes with acyl-CoA reductase (aldehyde forming) activity and aldehydereductase or alcohol dehydrogenase activities. Exemplary bifunctionaloxidoreductases that convert an acyl-CoA to alcohol are describedelsewhere herein.

FIG. 6 shows pathways for converting 1,3-butanediol to 3-buten-2-oland/or butadiene. Enzymes in FIG. 6 are A. 1,3-butanediol kinase, B.3-hydroxybutyrylphosphate kinase, C. 3-hydroxybutyryldiphosphate lyase,D. 1,3-butanediol diphosphokinase, E. 1,3-butanediol dehydratase, F.3-hydroxybutyrylphosphate lyase, G. 3-buten-2-ol dehydratase or chemicalreaction.

A. 1,3-Butanediol Kinase.

Phosphorylation of 1,3-butanediol to 3-hydroxybutyrylphosphate iscatalyzed by an alcohol kinase enzyme. Alcohol kinase enzymes catalyzethe transfer of a phosphate group to a hydroxyl group. Kinases thatcatalyze transfer of a phosphate group to an alcohol group are membersof the EC 2.7.1 enzyme class. The table herein in the section on CrotylAlcohol Kinase lists several useful kinase enzymes in the EC 2.7.1enzyme class.

Mevalonate kinase (EC 2.7.1.36) phosphorylates the terminal hydroxylgroup of mevalonate. Gene candidates for this step include erg12 from S.cerevisiae, mvk from Methanocaldococcus jannaschi, MVK from Homosapiens, and mvk from Arabidopsis thaliana col. Additional mevalonatekinase candidates include those described herein in the section onCrotyl Alcohol Kinase.

Glycerol kinase also phosphorylates the terminal hydroxyl group inglycerol to Ellin glycerol-3-phosphate. Additional glycerol kinasecandidates include those described herein in the section on CrotylAlcohol Kinase. Homoserine kinase is another similar enzyme candidate.Additional homoserine kinase candidates include those described hereinin the section on Crotyl Alcohol Kinase.

B. 3-Hydroxybutyrylphosphate Kinase.

Alkyl phosphate kinase enzymes catalyze the transfer of a phosphategroup to the phosphate group of an alkyl phosphate. The enzymesdescribed herein and in the section for 2-Butenyl-4-phosphate Kinasenaturally possess such activity or can be engineered to exhibit thisactivity, and include several useful kinase enzymes in the EC 2.7.4enzyme class.

Phosphomevalonate kinase enzymes are of particular interest.Phosphomevalonate kinase (EC 2.7.4.2) catalyzes the phosphorylation ofphosphomevalonate. Exemplary candidates include those listed herein inthe section 2-Butenyl-4-phosphate Kinase.

Famesyl monophosphate kinase enzymes catalyze the CTP dependentphosphorylation of famesyl monophosphate to famesyl diphosphate.Similarly, geranylgeranyl phosphate kinase catalyzes CTP dependentphosphorylation. Enzymes with these activities were identified in themicrosomal fraction of cultured Nicotiana tabacum (Thai et al, PNAS96:13080-5 (1999)). However, the associated genes have not beenidentified to date.

C. 3-Hydroxybutyryldiphosphate Lyase.

Diphosphate lyase enzymes catalyze the conversion of alkyl diphosphatesto alkenes. Carbon-oxygen lyases that operate on phosphates are found inthe EC 4.2.3 enzyme class. The table below lists several useful enzymesin EC class 4.2.3. described herein. Exemplary enzyme candidates alsoinclude phosphate lyases described herein.

Enzyme Commission No. Enzyme Name 4.2 3.5 Chorismate synthase 4.2.3.15Myrcene synthase 4.2.3.27 Isoprene synthase 4.2.3.36 Terpentrienesythase 4.2.3.46 (E, E)-alpha-Farnesene synthase 4.2.3.47 Beta-Farnesenesynthase

D. 1,3-Butanediol Dehydratase.

Exemplary dehydratase enzymes suitable for dehydrating 1,3-butanediol to3-buten-2-ol include oleate hydratases, acyclic 1,2-hydratases andlinalool dehydratase. Exemplary enzyme candidates are described above.

E. 1,3-Butanediol Diphosphokinase.

Diphosphokinase enzymes catalyze the transfer of a diphosphate group toan alcohol group. The enzymes described in the section on Crotyl AlcoholDiphosphokinase naturally possess such activity. Kinases that catalyzetransfer of a diphosphate group are members of the EC 2.7.6 enzymeclass.

Of particular interest are ribose-phosphate diphosphokinase enzymes,also described in the section on Crotyl Alcohol Diphosphokinase.

F. 3-Hydroxybutyrylphosphate Lyase.

Phosphate lyase enzymes catalyze the conversion of alkyl phosphates toalkenes. Carbon-oxygen lyases that operate on phosphates are found inthe EC 4.2.3 enzyme class. The section herein on Butadiene Synthase(monophosphate) enzymesdescribes relevant enzymes in EC class 4.2.3.

Isoprene synthase enzymes catalyzes the conversion of dimethylallyldiphosphate to isoprene and suitable enzymes are described in thesection on Butadiene Synthase (monophosphate).

Chorismate synthase (EC 4.2.3.5) participates in the shikimate pathway,catalyzing the dephosphorylation of 5-enolpyruvylshikimate-3-phosphateto chorismate and suitable enzymes are described in the section onButadiene

Synthase (monophosphate).

Myrcene synthase enzymes catalyze the dephosphorylation of geranyldiphosphate to beta-myrcene (EC 4.2.3.15). Exemplary myrcene synthasesare described in the section on Butadiene Synthase (monophosphate).

Famesyl diphosphate is converted to alpha-famesene and beta-famesene byalpha-famesene synthase and beta-famesene synthase, respectively.Exemplary alpha-famesene synthase enzymes include those described in thesection on Butadiene Synthase (monophosphate).

G. G. 3-Buten-2-ol Dehydratase.

Dehydration of 3-buten-2-ol to butadiene is catalyzed by a 3-buten-2-oldehydratase enzyme or by chemical dehydration. Exemplary dehydrataseenzymes suitable for dehydrating 3-buten-2-ol include oleate hydratase,acyclic 1,2 hydratase and linalool dehydratase enzymes. Exemplaryenzymes are described above.

Example VIII 1,4-Butanediol Synthesis Enzymes

This Example provides genes that can be used for conversion ofsuccinyl-CoA to 1,4-butanediol as depicted in the pathways of FIG. 7.

A) Succinyl-CoA Transferase (designated as EB1) or Succinyl-CoASynthetase (designated as EB2A).

The conversion of succinate to succinyl-CoA is catalyzed by EB1 or EB2A(synthetase or ligase). Exemplary EB1 and EB2A enzymes are describedabove.

B) Succinyl-CoA Reductase (Aldehyde Forming).

Enzymes with succinyl-CoA reductase activity are encoded by sucD ofClostridium kluyveri (Sohling, J. Bacteriol. 178:871-880 (1996)) andsucD of Porphyromonas gingivalis (Takahashi, J. Bacteriol 182:4704-4710(2000)). Additional succinyl-CoA reductase enzymes participate in the3-hydroxypropionate/4-HB cycle of thermophilic archaea such asMetallosphaera sedula (Berg et al., Science 318:1782-1786 (2007)) andThermoproteus neutrophilus (Ramos-Vera et al., J Bacteriol,191:4286-4297 (2009)). These and other exemplary succinyl-CoA reductaseenzymes are described above.

C) 4-HB Dehydrogenase (Designated as EB4).

Enzymes exhibiting EB4 activity (EC 1.1.1.61) have been characterized inRalstonia eutropha (Bravo et al., J. Forensic Sci. 49:379-387 (2004),Clostridium kluyveri (Wolff and Kenealy, Protein Expr. Purif. 6:206-212(1995)) and Arabidopsis thaliana (Breitkreuz et al., J. Biol. Chem.278:41552-41556 (2003)). Other EB4 enzymes are found in Porphyromonasgingivalis and gbd of an uncultured bacterium. Accession numbers ofthese genes are listed in the table below.

Protein GenBank ID GI Number Organism 4hbd YP_726053.1 113867564Ralstonia eutropha H16 4hbd L21902.1 146348486 Clostridium kluyveri DSM555 4hbd Q94B07 75249805 Arabidopsis thaliana 4-hBd NP_904964.1 34540485Porphyromonas gingivalis W83 gbd AF148264.1 5916168 Uncultured bacterium

D) 4-HB Kinase (Designated as EB5).

Activation of 4-HB to 4-hydroxybutyryl-phosphate is catalyzed by EBS.Phosphotransferase enzymes in the EC class 2.7.2 transform carboxylicacids to phosphonic acids with concurrent hydrolysis of one ATP. Enzymessuitable for catalyzing this reaction include butyrate kinase, acetatekinase, aspartokinase and gamma-glutamyl kinase. Other butyrate kinaseenzymes are found in C. butyricum, C. beijerinckii and C. tetanomorphum(Twarog and Wolfe, J. Bacteriol. 86:112-117 (1963)). Aspartokinasecatalyzes the ATP-dependent phosphorylation of aspartate andparticipates in the synthesis of several amino acids. The aspartokinaseIII enzyme in E. coli, encoded by lysC, has a broad substrate range, andthe catalytic residues involved in substrate specificity have beenelucidated. Exemplary enzymes are below.

Gene Accession No. GI No. Organism buk1 NP_349675 15896326 Clostridiumacetobutylicum buk2 Q97II1 20137415 Clostridium acetobutylicum buk2Q9X278.1 6685256 Thermotoga maritima lysC NP_418448.1 16131850Escherichia coli ackA NP_416799.1 16130231 Escherichia coli proBNP_414777.1 16128228 Escherichia coli buk YP_001307350.1 150015096Clostridium beijerinckii buk2 YP_001311072.1 150018818 Clostridiumbeijerinckii

E) Phosphotrans-4-Hydroxybutyrylase (Designated as EB6).

EB6 catalyzes the transfer of the 4-hydroxybutyryl group from phosphateto CoA. Acyltransferases suitable for catalyzing this reaction includephosphotransacetylase and phosphotransbutyrylase. The pta gene from E.coli encodes an enzyme that can convert acetyl-phosphate into acetyl-CoA(Suzuki, Biochim. Biophys. Acta 191:559-569 (1969)). This enzyme canalso utilize propionyl-CoA instead of acetyl-CoA (Hesslinger et al.,Mol. Microbiol 27:477-492 (1998)). Similarly, the ptb gene from C.acetobutylicum encodes an enzyme that can convert butyryl-CoA intobutyryl-phosphate (Walter et al., Gene 134:107-111 (1993)); Huang etal., J Mol. Microbiol. Biotechno.l 2:33-38 (2000). Additional ptb genescan be found in Clostridial organisms, butyrate producing bacteriumL2-50 (Louis et al., J. Bacteriol. 186:2099-2106 (2004)) and Bacillusmegaterium (Vazquez et al., Curr. Microbiol. 42:345-349 (2001)).

Gene Accession No. GI No. Organism pta NP_416800.1 16130232 Escherichiacoli ptb NP_349676 15896327 Clostridium acetobutylicum ptbYP_001307349.1 150015095 Clostridium beijerinckii ptb AAR19757.138425288 butyrate-producing bacterium L2-50 ptb CAC07932.1 10046659Bacillus megaterium

F) 4-Hydroxybutyryl-CoA Reductase (Aldehyde Forming).

Enzymes with this activity are described above.

G) 1,4-Butanediol Dehydrogenase (Designated as EB8).

EB8 catalyzes the reduction of 4-hydroxybutyraldehyde to 1,4-butanediol.Enzymes with 1,4-butanediol activity are listed in the table below.

Protein GenBank ID GI Number Organism alrA BAB12273.1 9967138Acinetobacter sp. strain M-1 ADH2 NP_014032.1 6323961 Saccharomycescerevisiae fucO NP_417279.1 16130706 Escherichia coli yqhD NP_417484.116130909 Escherichia coli bdh I NP_349892.1 15896543 Clostridiumacetobutylicum bdh II NP_349891.1 15896542 Clostridium acetobutylicumbdh BAF45463.1 124221917 Clostridium saccharoperbutylacetonicumCbei_1722 YP_001308850 150016596 Clostridium beijerinckii Cbei_2181YP_001309304 150017050 Clostridium beijerinckii Cbei 2421 YP_001309535150017281 Clostridium beijerinckii 14bdh AAC76047.1 1789386 Escherichiacoli K-12 MG1655 14bdh YP_001309304.1 150017050 Clostridium beijerinckiiNCIMB 8052 14bdh P13604.1 113352 Clostridium saccharobutylicum 14bdhZP_03760651.1 225405462 Clostridium asparagiforme DSM 15981 14bdhZP_02083621.1 160936248 Clostridium bolteae ATCC BAA-613 14bdhYP_003845251.1 302876618 Clostridium cellulovorans 743B 14bdhZP_03294286.1 210624270 Clostridium hiranonis DSM 13275 14bdhZP_03705769.1 225016577 Clostridium methylpentosum DSM 5476 14bdhYP_003179160.1 257783943 Atopobium parvulum DSM 20469 14bdhYP_002893476.1 237809036 Tolumonas auensis DSM 9187 14bdh ZP_05394983.1255528157 Clostridium carboxidivorans P7

H) Succinate Reductase.

Direct reduction of succinate to succinate semialdehyde is catalyzed bya carboxylic acid reductase. Exemplary enzymes for catalyzing thistransformation are also those described below and herein for K)4-Hydroxybutyrate reductase.

I) Succinyl-CoA Reductase (Alcohol Forming) (Designated as EB10).

EB10 enzymes are bifunctional oxidoreductases that convert succinyl-CoAto 4-HB. Enzyme candidates described below and herein for M)4-hydroxybutyryl-CoA reductase (alcohol forming) are also suitable forcatalyzing the reduction of succinyl-CoA.

J) 4-Hydroxybutyryl-CoA Transferase or 4-Hydroxybutyryl-CoA Synthetase(Designated as EB11).

Conversion of 4-HB to 4-hydroxybutyryl-CoA is catalyzed by a CoAtransferase or synthetase. EB11 enzymes include those listed below.

Protein GenBank ID GI Number Organism cat1 P38946.1 729048 Clostridiumkluyveri cat2 P38942.2 172046066 Clostridium kluyveri cat3 EDK35586.1146349050 Clostridium kluyveri TVAG_395550 XP_001330176 123975034Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875 Trypanosomabrucei cat2 CAB60036.1 6249316 Clostridium aminobutyricum cat2NP_906037.1 34541558 Porphyromonas gingivalis W83

4HB-CoA synthetase catalyzes the ATP-dependent conversion of 4-HB to4-hydroxybutyryl-CoA. AMP-forming 4-HB-CoA synthetase enzymes are foundin organisms that assimilate carbon via thedicarboxylate/hydroxybutyrate cycle or the 3-hydroxypropionate/4-HBcycle. Enzymes with this activity have been characterized inThermoproteus neutrophilus and Metallosphaera sedula. Others can beinferred by sequence homology. ADP forming CoA synthetases, such EB2A,are also suitable candidates.

Protein GenBank ID GI Number Organism Tneu 0420 ACB39368.1 170934107Thermoproteus neutrophilus Caur_0002 YP_001633649.1 163845605Chloroflexus aurantiacus J-10-fl Cagg 3790 YP_002465062 219850629Chloroflexus aggregans DSM 9485 acs YP_003431745 288817398Hydrogenobacter thermophilus TK-6 Pisl 0250 YP_929773.1 119871766Pyrobaculum islandicum DSM 4184 Msed 1422 ABP95580.1 145702438Metallosphaera sedula

K) 4-HB Reductase.

Reduction of 4-HB to 4-hydroxybutanal is catalyzed by a carboxylic acidreductase (CAR) such as the Car enzyme found in Nocardia iowensis. Thisenzyme and other carboxylic acid reductases are described above (see EC1.2.1.e).

L) 4-Hydroxybutyryl-Phosphate Reductase (Designated as EB14).

EB14 catalyzes the reduction of 4-hydroxybutyrylphosphate to4-hydroxybutyraldehyde. An enzyme catalyzing this transformation has notbeen identified to date. However, similar enzymes include phosphatereductases in the EC class 1.2.1. Exemplary phosphonate reductaseenzymes include G3P dehydrogenase (EC 1.2.1.12), aspartate-semialdehydedehydrogenase (EC 1.2.1.11) acetylglutamylphosphate reductase (EC1.2.1.38) and glutamate-5-semialdehyde dehydrogenase (EC 1.2.1.-).Aspartate semialdehyde dehydrogenase (ASD, EC 1.2.1.11) catalyzes theNADPH-dependent reduction of 4-aspartyl phosphate toaspartate-4-semialdehyde. The E. coli ASD enzyme has been shown toaccept the alternate substrate beta-3-methylaspartyl phosphate (Shameset al., J Biol. Chem. 259:15331-15339 (1984)). A related enzymecandidate is acetylglutamylphosphate reductase (EC 1.2.1.38), an enzymethat naturally reduces acetylglutamylphosphate toacetylglutamate-5-semialdehyde, found in S. cerevisiae (Pauwels et al.,Eur. J Biochem. 270:1014-1024 (2003)), B. subtilis (O'Reilly et al.,Microbiology 140 (Pt 5):1023-1025 (1994)), E. coli (Parsot et al., Gene.68:275-283 (1988)), and other organisms. Additional phosphate reductaseenzymes of E. coli include glyceraldehyde 3-phosphate dehydrogenase(gapA (Branlant et al., Eur. J. Biochem. 150:61-66 (1985))) andglutamate-5-semialdehyde dehydrogenase (proA (Smith et al., J.Bacteriol. 157:545-551 (1984))). Genes encoding glutamate-5-semialdehydedehydrogenase enzymes from Salmonella typhimurium (Mahan et al., JBacteria 156:1249-1262 (1983)) and Campylobacter jejuni (Louie et al.,Mol. Gen. Genet. 240:29-35 (1993)) were cloned and expressed in E. coli.

Protein GenBank ID GI Number Organism asd NP_417891.1 16131307Escherichia coli asd YP_248335.1 68249223 Haemophilus influenzae asdAAB49996 1899206 Mycobacterium tuberculosis VC2036 NP_231670 15642038Vibrio cholera asd YP_002301787.1 210135348 Heliobacter pylori ARG5,6NP_010992.1 6320913 Saccharomyces cerevisiae argC NP_389001.1 16078184Bacillus subtilis argC NP_418393.1 16131796 Escherichia coli gapAP0A9B2.2 71159358 Escherichia coli proA NP_414778.1 16128229 Escherichiacoli proA NP_459319.1 16763704 Salmonella typhimurium proA P53000.29087222 Campylobacter jejuni

M) 4-Hydroxybutyryl-CoA Reductase (Alcohol Forming) (Designated asEB15).

EB15 enzymes are bifunctional oxidoreductases that convert an4-hydroxybutyryl-CoA to 1,4-butanediol. Enzymes with this activityinclude those below.

Protein GenBank ID GI Number Organism adhE NP_415757.1 16129202Escherichia coli adhE2 AAK09379.1 12958626 Clostridium acetobutylicumbdh I NP_349892.1 15896543 Clostridium acetobutylicum bdh II NP_349891.115896542 Clostridium acetobutylicum adhE AAV66076.1 55818563 Leuconostocmesenteroides adhE NP_781989.1 28211045 Clostridium tetani adhENP_563447.1 18311513 Clostridium perfringens adhE YP_001089483.1126700586 Clostridium difficile

Example IX Adipate, 6-Aminocaproate, Caprolactam andHexamethylenediamine Synthesis Enzymes

This Example provides genes that can be used for conversion ofsuccinyl-CoA and acetyl-CoA to adipate, 6-aminocaproate, caprolactam andhexamethylenediamine as depicted in the pathways of FIG. 8.

FIG. 8. depicts enzymes: A) 3-oxoadipyl-CoA thiolase, B) 3-oxoadipyl-CoAreductase, C) 3-hydroxyadipyl-CoA dehydratase, D)5-carboxy-2-pentenoyl-CoA reductase, E) adipyl-CoA reductase (aldehydeforming), F) 6-aminocaproate transaminase, or 6-aminocaproatedehydrogenase, G) 6-aminocaproyl-CoA/acyl-CoA transferase, or6-aminocaproyl-CoA synthase, H) amidohydrolase, I) spontaneouscyclization, J) 6-aminocaproyl-CoA reductase (aldehyde forming), K) HMDAtransaminase or HMDA dehydrogenase, L) Adipyl-CoA hydrolase, adipyl-CoAligase, adipyl-CoA transferase, or phosphotransadipylase/adipate kinase.

Transformations depicted in FIG. 8 fall into at least 10 generalcategories of transformations shown in the Table below. The first threedigits of each label correspond to the first three Enzyme Commissionnumber digits which denote the general type of transformationindependent of substrate specificity. Below is described a number ofbiochemically characterized candidate genes in each category.Specifically listed are exemplary genes that can be applied to catalyzethe appropriate transformations in FIG. 8 when cloned and expressed.

Step Label Function FIG. 8, step B 1.1.1.a Oxidoreductase (ketone tohydroxyl or aldehyde to alcohol) FIG. 8, steps E 1.2.1.b Oxidoreductase(acyl-CoA to and J aldehyde) FIG. 8, step D 1.3.1.a Oxidoreductaseoperating on CH—CH donors FIG. 8, steps F 1.4.1.a Oxidoreductaseoperating on and K amino acids FIG. 8, step A 2.3.1.b AcyltransferaseFIG. 8, steps F 2.6.1.a Aminotransferase and K FIG. 8, steps G 2.8.3.aCoenzyme-A transferase and L FIG. 8, steps G 6.2.1.a Acid-thiol ligaseand L FIG. 8, Step H 6.3.1.a/ Amide synthases/peptide 6.3.2.a synthasesFIG. 8, step I No enzyme Spontaneous cyclization required

FIG. 8, Step A—3-Oxoadipyl-CoA Thiolase.

EC 2.3.1.b Acyl Transferase.

The first step in the pathway combines acetyl-CoA and succinyl-CoA toform 3-oxoadipyl-CoA. Step A can involve a 3-oxoadipyl-CoA thiolase, orequivalently, succinyl CoA:acetyl CoA acyl transferase (β-ketothiolase).Since beta-ketothiolase enzymes catalyze reversible transformations,these enzymes can be employed for the synthesis of 3-oxoadipyl-CoA. Forexample, the ketothiolase phaA from R. eutropha combines two moleculesof acetyl-CoA to form acetoacetyl-CoA (Sato et al., J Biosci Bioeng103:38-44 (2007)). Similarly, a β-keto thiolase (bktB) has been reportedto catalyze the condensation of acetyl-CoA and propionyl-CoA to formβ-ketovakryl-CoA (Slater et al., J. Bacteriol. 180:1979-1987 (1998)) inR. eutropha. The protein sequences for the above-mentioned gene productsare well known in the art and can be accessed in the public databasessuch as GenBank using the following accession numbers.

Gene name GI Number GenBank ID Organism paaJ 16129358 NP_415915.1Escherichia coli pcaF 17736947 AAL02407 Pseudomonas knackmussii (B13)phaD 3253200 AAC24332.1 Pseudomonas putida paaE 106636097 ABF82237.1Pseudomonas fluorescens

These exemplary sequences can be used to identify homologue proteins inGenBank or other databases through sequence similarity searches (forexample, BLASTp). The resulting homologue proteins and theircorresponding gene sequences provide additional exogenous DNA sequencesfor transformation into E. coli or other suitable host microorganisms togenerate production hosts.

For example, orthologs of paaJ from Escherichia coli K12 can be foundusing the following GenBank accession numbers:

GI Number GenBank ID Organism 152970031 YP_001335140.1 Klebsiellapneumoniae 157371321 YP_001479310.1 Serratia proteamaculans 3253200AAC24332.1 Pseudomonas putida

Example orthologs of pcaF from Pseudomonas knackmussii can be foundusing the following GenBank accession numbers:

GI Number GenBank ID Organism 4530443 AAD22035.1 Streptomyces sp. 206524982839 AAN67000.1 Pseudomonas putida 115589162 ABJ15177.1 Pseudomonasaeruginosa

Additional native candidate genes for the ketothiolase step includeatoB, which can catalyze the reversible condensation of 2 acetyl-CoAmolecules (Sato et al., J. Biosci. Bioengineer. 103:38-44 (2007)), andits homolog yqeF. Non-native gene candidates include those in the tablebelow. The protein sequences for each of these exemplary gene productscan be found using the following GenBank accession numbers:

Gene Name GenBank ID Organism atoB NP_416728.1 Escherichia coli yqeFNP_417321.2 Escherichia coli phaA YP_725941 Ralstonia eutropha bktBAAC38322.1 Ralstonia eutropha thiA NP_349476.1 Clostridiumacetobutylicum thiB NP_149242.1 Clostridium acetobutylicum

2-Amino-4-oxopentanoate (AKP) thiolase or AKP thiolase (AKPT) enzymespresent additional candidates for performing step A. AKPT is a pyridoxalphosphate-dependent enzyme participating in omithine degradation inClostridium sticklandii (Jeng et al., Biochemistry 13:2898-2903 (1974);Kenklies et al., Microbiology 145:819-826 (1999)). A gene clusterencoding the alpha and beta subunits of AKPT (or-2 (ortA) and or-3(ortB)) was recently identified and the biochemical properties of theenzyme were characterized (Fonknechten et al., J. Bacteriol. In Press(2009)). The enzyme is capable of operating in both directions andnaturally reacts with the D-isomer of alanine. AKPT from Clostridiumsticklandii has been characterized but its protein sequence has not yetbeen published. Enzymes with high sequence homology are found inClostridium difficile, Alkaliphilus metalliredigenes QYF,Thermoanaerobacter sp. X514, and Thermoanaerobacter tengcongensis MB4(Fonknechten et al., supra).

Gene name GI Number GenBank ID Organism ortA (α) 126698017YP_001086914.1 Clostridium difficile 630 ortB (β) 126698018YP_001086915.1 Clostridium difficile 630 Amet_2368 (α) 150390132YP_001320181.1 Alkaliphilus metalliredigenes QYF Amet_2369 (β) 150390133YP_001320182.1 Alkaliphilus metalliredigenes QYF Teth514_1478 (α)167040116 YP_001663101.1 Thermoanaerobacter sp. X514 Teth514_1479 (β)167040117 YP_001663102.1 Thermoanaerobacter sp. X514 TTE1235 (α)20807687 NP_622858.1 Thermoanaerobacter tengcongensis MB4 thrC (β)20807688 NP_622859.1 Thermoanaerobacter tengcongensis MB4

Step B—3-Oxoadipyl-CoA Reductase.

EC 1.1.1.a Oxidoreductases.

Certain transformations depicted in FIG. 8 involve oxidoreductases thatconvert a ketone functionality to a hydroxyl group. For example, FIG. 8,step B involves the reduction of a 3-oxoacyl-CoA to a 3-hydroxyacyl-CoA.

Exemplary enzymes that can convert 3-oxoacyl-CoA molecules, such as3-oxoadipyl-CoA, into 3-hydroxyacyl-CoA molecules, such as3-hydroxyadipyl-CoA, include enzymes whose natural physiological rolesare in fatty acid beta-oxidation or phenylacetate catabolism. Forexample, subunits of two fatty acid oxidation complexes in E. coli,encoded by fadB and fadJ, function as 3-hydroxyacyl-CoA dehydrogenases(Binstock et al., Methods Enzymol. 71:403-411 (1981)). Furthermore, thegene products encoded by phaC in Pseudomonas putida U (Olivera et al.,Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)) and paaC in Pseudomonasfluorescens ST (Di Gennaro et al., Arch. Microbiol. 188:117-125 (2007))catalyze the reverse reaction of step B in FIG. 8, that is, theoxidation of 3-hydroxyadipyl-CoA to form 3-oxoadipyl-CoA, during thecatabolism of phenylacetate or styrene. Note that the reactionscatalyzed by such enzymes are reversible. A similar transformation isalso carried out by the gene product of hbd in Clostridiumacetobutylicum (Atsumi et al., Metab. Eng. (epub Sep. 14, 2007); Boyntonet al., J. Bacteriol. 178:3015-3024 (1996)). This enzyme convertsacetoacetyl-CoA to 3-hydroxybutyryl-CoA. In addition, given theproximity in E. coli of paaH to other genes in the phenylacetatedegradation operon (Nogales et al., Microbiology 153:357-365 (2007)) andthe fact that paaH mutants cannot grow on phenylacetate (Ismail et al.,Eur. J Biochem. 270:3047-3054 (2003)), it is expected that the E. colipaaH gene encodes a 3-hydroxyacyl-CoA dehydrogenase.

Gene name GI Number GenBank ID Organism fadB 119811 P21177.2 Escherichiacoli fadJ 3334437 P77399.1 Escherichia coli paaH 16129356 NP_415913.1Escherichia coli phaC 26990000 NP_745425.1 Pseudomonas putida paaC106636095 ABF82235.1 Pseudomonas fluorescens

Additional exemplary oxidoreductases capable of converting 3-oxoacyl-CoAmolecules to their corresponding 3-hydroxyacyl-CoA molecules include3-hydroxybutyryl-CoA dehydrogenases. Exemplary enzymes are shown below.

Gene name GI Number GenBank ID Organism hbd 18266893 P52041.2Clostridium acetobutylicum Hbd2 146348271 EDK34807.1 Clostridiumkluyveri Hbd1 146345976 EDK32512.1 Clostridium kluyveri HSD17B10 3183024O02691.3 Bos taurus phbB 130017 P23238.1 Zoogloea ramigera phaB146278501 YP_001168660.1 Rhodobacter sphaeroides

A number of similar enzymes have been found in other species ofClostridia and in Metallosphaera sedula (Berg et al., Science318:1782-1786 (2007)).

Gene name GI Number GenBank ID Organism hbd 15895965 NP_349314.1Clostridium acetobutylicum hbd 20162442 AAM14586.1 Clostridiumbeijerinckii Msed 1423 146304189 YP_001191505 Metallosphaera sedulaMsed_0399 146303184 YP_001190500 Metallosphaera sedula Msed_0389146303174 YP_001190490 Metallosphaera sedula Msed_1993 146304741YP_001192057 Metallosphaera sedula

Step C—3-Hydroxyadipyl-CoA Dehydratase.

Step C can involve a 3-hydroxyadipyl-CoA dehydratase. The gene productof crt from C. acetobutylicum catalyzes the dehydration of3-hydroxybutyryl-CoA to crotonyl-CoA (Atsumi et al., Metab. Eng. (epubSep. 14, 2007); Boynton et al., J. Bacteriol. 178:3015-3024 (1996)).Homologs of this gene are strong candidates for carrying out the thirdstep (step C) in the synthesis pathways exemplified in FIG. 8. Inaddition, genes known to catalyze the hydroxylation of double bonds inenoyl-CoA compounds represent additional candidates given thereversibility of such enzymatic transformations. For example, theenoyl-CoA hydratases, phaA and phaB, of P. putida are believed to carryout the hydroxylation of double bonds during phenylacetate catabolism(Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)) andthus represent additional candidates for incorporation into E. coli.Lastly, a number of Escherichia coli genes have been shown todemonstrate enoyl-CoA hydratase functionality including maoC (Park andLee, J. Bacteriol. 185:5391-5397 (2003)), paaF (Ismail et al., Eur. J.Biochem. 270:3047-3054 (2003); Park and Lee, Biotechnol. Bioeng.86:681-686 (2004); Park and Lee, Appl. Biochem. Biotechnol.113-116:335-346 (2004)), and paaG (Ismail et al., supra, 2003; Park andLee, supra, 2003; Park and Lee, supra, 2004). The protein sequences foreach of these exemplary gene products can be found using the followingGenBank accession numbers:

Gene Name GenBank ID Organism maoC NP_415905.1 Escherichia coli paaFNP_415911.1 Escherichia coli paaG NP_415912.1 Escherichia coli crtNP_349318.1 Clostridium acetobutylicum paaA NP_745427.1 Pseudomonasputida paaB NP_745426.1 Pseudomonas putida phaA ABF82233.1 Pseudomonasfluorescens phaB ABF82234.1 Pseudomonas fluorescens

Alternatively, beta-oxidation genes are candidates for the first threesteps in adipate synthesis. Candidate genes for the proposed adipatesynthesis pathway also include the native fatty acid oxidation genes ofE. coli and their homologs in other organisms. The E. coli genes fadAand fadB encode a multienzyme complex that exhibits ketoacyl-CoAthiolase, 3-hydroxyacyl-CoA dehydrogenase, and enoyl-CoA hydrataseactivities (Yang et al., Biochem. 30:6788-6795 (1991); Yang et al., J.Biol. Chem. 265:10424-10429 (1990); Yang et al., J. Biol. Chem.266:16255 (1991); Nakahigashi and Inokuchi, Nucl. Acids Res. 18: 4937(1990)). These activities are mechanistically similar to the first threetransformations shown in FIG. 8. The fadI and fadJ genes encode similarfunctions and are naturally expressed only anaerobically (Campbell etal., Mol. Microbiol. 47:793-805 (2003)). These gene products naturallyoperate to degrade short, medium, and long chain fatty-acyl-CoAcompounds to acetyl-CoA, rather than to convert succinyl-CoA andacetyl-CoA into 5-carboxy-2-pentenoyl-CoA as proposed in FIG. 8.However, it is well known that the ketoacyl-CoA thiolase,3-hydroxyacyl-CoA dehydrogenase, and enoyl-CoA hydratase enzymescatalyze reversible transformations. Furthermore, directed evolution andrelated approaches can be applied to tailor the substrate specificitiesof the native beta-oxidation machinery of E. coli. Thus these enzymes orhomologues thereof can be applied for adipate production. If the nativegenes operate to degrade adipate or its precursors in vivo, theappropriate genetic modifications are made to attenuate or eliminatethese functions. However, it may not be necessary since a method forproducing poly[(R)-3-hydroxybutyrate] in E. coli that involvesactivating fadB, by knocking out a negative regulator, fadR, andco-expressing a non-native ketothiolase, phaA from Ralstonia eutropha,has been described (Sato et al., J. Biosci. Bioeng. 103:38-44 (2007)).This work clearly demonstrated that a beta-oxidation enzyme, inparticular the gene product of fadB which encodes both 3-hydroxyacyl-CoAdehydrogenase and enoyl-CoA hydratase activities, can function as partof a pathway to produce longer chain molecules from acetyl-CoAprecursors. The protein sequences for each of these exemplary geneproducts can be found using the following GenBank accession numbers:

Gene Name GenBank ID Organism fadA YP_026272.1 Escherichia coli fadBNP_418288.1 Escherichia coli fadI NP_416844.1 Escherichia coli fadJNP_416843.1 Escherichia coli fadR NP_415705.1 Escherichia coli

Step D—5-Carboxy-2-Pentenoyl-CoA Reductase. EC 1.3.1.a OxidoreductaseOperating on CH—CH Donors.

Step D involves the conversion of 5-carboxy-2-pentenoyl-CoA toadipyl-CoA by 5-carboxy-2-pentenoyl-CoA reductase. Enoyl-CoA reductaseenzymes are suitable enzymes for this transformation.

Whereas the ketothiolase, dehydrogenase, and enoyl-CoA hydratase stepsare generally reversible, the enoyl-CoA reductase step is almost alwaysoxidative and irreversible under physiological conditions (Hollmeisteret al., Biol. Chem. 280:4329-4338 (2005)). FadE catalyzes this likelyirreversible transformation in E. coli (Campbell and Cronan, JBacteriol. 184:3759-3764 (2002)). The pathway can involve an enzyme thatreduces a 2-enoyl-CoA intermediate, not one such as FadE that will onlyoxidize an acyl-CoA to a 2-enoyl-CoA compound. Furthermore, although ithas been suggested that E. coli naturally possesses enzymes forenoyl-CoA reduction (Mizugaki et al., Biochem. 92:1649-1654 (1982);Nishimaki et al., J Biochem. 95:1315-1321 (1984)), no E. coli genepossessing this function has been biochemically characterized.

One exemplary enoyl-CoA reductase is the gene product of bcd from C.acetobutylicum (Boynton et al., J Bacteriol. 178:3015-3024 (1996);Atsumi et al., Metab. Eng. 2008 10(6):305-311 (2008)(Epub Sep. 14,2007), which naturally catalyzes the reduction of crotonyl-CoA tobutyryl-CoA. Activity of this enzyme can be enhanced by expressing bcdin conjunction with expression of the C. acetobutylicum etfAB genes,which encode an electron transfer flavoprotein. An additional candidatefor the enoyl-CoA reductase step is the mitochondrial enoyl-CoAreductase from E. gracilis (Hoffmeister et al., J. Biol. Chem.280:4329-4338 (2005)). A construct derived from this sequence followingthe removal of its mitochondrial targeting leader sequence was cloned inE. coli resulting in an active enzyme (Hoffmeister et al., supra). Thisapproach is well known to those skilled in the art of expressingeukaryotic genes, particularly those with leader sequences that maytarget the gene product to a specific intracellular compartment, inprokaryotic organisms. A close homolog of this gene, TDE0597, from theprokaryote Treponema denticola represents a third enoyl-CoA reductasewhich has been cloned and expressed in E. coli (Tucci et al., FEBSLetters 581:1561-1566 (2007)).

Gene name GI Number GenBank ID Organism bcd 15895968 NP_349317.1Clostridium acetobutylicum etfA 15895966 NP_349315.1 Clostridiumacetobutylicum etfB 15895967 NP_349316.1 Clostridium acetobutylicum TER62287512 Q5EU90.1 Euglena gracilis TDE0597 42526113 NP_971211.1Treponema denticola

Step E—Adipyl-CoA Reductase (Aldehyde Forming). EC 1.2.1.bOxidoreductase (Acyl-CoA to Aldehyde).

The transformation of adipyl-CoA to adipate semialdehyde in step E caninvolve an acyl-CoA dehydrogenases capable of reducing an acyl-CoA toits corresponding aldehyde. An EC 1.2.1.b oxidoreductase (acyl-CoA toaldehyde) provides suitable enzyme activity. Exemplary enzymes in thisclass are described above.

Step F—6-Aminocaproate Transaminase or 6-Aminocaproate Dehydrogenase. EC1.4.1.a Oxidoreductase Operating on Amino Acids.

Step F depicts a reductive amination involving the conversion of adipatesemialdehyde to 6-aminocaproate.

Most oxidoreductases operating on amino acids catalyze the oxidativedeamination of alpha-amino acids with NAD+ or NADP+ as acceptor, thoughthe reactions are typically reversible. Exemplary oxidoreductasesoperating on amino acids include glutamate dehydrogenase (deaminating),encoded by gdhA, leucine dehydrogenase (deaminating), encoded by ldh,and aspartate dehydrogenase (deaminating), encoded by nadX. The gdhAgene product from Escherichia coli (McPherson et al., Nucleic. AcidsRes. 11:5257-5266 (1983); Korber et al., J. Mol. Biol. 234:1270-1273(1993)), gdh from Thermotoga maritima (Kort et al., Extremophiles1:52-60 (1997); Lebbink et al., J. Mol. Biol. 280:287-296 (1998);Lebbink et al., J. Mol. Biol. 289:357-369 (1999)), and gdhA1 fromHalobacterium salinarum (Ingoldsby et al., Gene. 349:237-244 (2005))catalyze the reversible interconversion of glutamate to 2-oxoglutarateand ammonia, while favoring NADP(H), NAD(H), or both, respectively.Exemplary enzymes are described in the table below.

Gene name GI Number GenBank ID Organism gdhA 118547 P00370 Escherichiacoli gdh 6226595 P96110.4 Thermotoga maritima gdhA1 15789827 NP_279651.1Halobacterium salinarum ldh 61222614 P0A393 Bacillus cereus nadX15644391 NP_229443.1 Thermotoga maritima

The lysine 6-dehydrogenase (deaminating), encoded by the lysDHgenes,catalyze the oxidative deamination of the epsilon-amino group ofL-lysine to form 2-aminoadipate-6-semialdehyde, which in turnnonenzymatically cyclizes to form Δ¹-piperideine-6-carboxylate (Misonoet al., J. Bacteriol. 150:398-401 (1982)). Exemplary enzymes are shownin the table below. Such enzymes are particularly good candidates forconverting adipate semialdehyde to 6-aminocaproate given the structuralsimilarity between adipate semialdehyde and2-aminoadipate-6-semialdehyde.

Gene name GI Number GenBank ID Organism lysDH 13429872 BAB39707Geobacillus stearothermophilus lysDH 15888285 NP_353966 Agrobacteriumtumefaciens lysDH 74026644 AAZ94428 Achromobacter denitrificans

EC 2.6.1.a Aminotransferase.

Step F of FIG. 8 can also, in certain embodiments, involve thetransamination of a 6-aldehyde to an amine. This transformation can becatalyzed by gamma-aminobutyrate transaminase (GABA transaminase).Exemplary enzymes are shown in the table below.

Gene name GI Number GenBank ID Organism gabT 16130576 NP_417148.1Escherichia coli puuE 16129263 NP_415818.1 Escherichia coli abat37202121 NP_766549.2 Mus musculus gabT 70733692 YP_257332.1 Pseudomonasfluorescens abat 47523600 NP_999428.1 Sus scrofa

Additional enzyme candidates include putrescine aminotransferases orother diamine aminotransferases. Such enzymes are particularly wellsuited for carrying out the conversion of 6-aminocaproate semialdehydeto hexamethylenediamine. The E. coli putrescine aminotransfemse isencoded by the ygjG gene and the purified enzyme also was able totransaminate cadaverine and spermidine (Samsonova et al., BMC Microbiol3:2 (2003)). In addition, activity of this enzyme on 1,7-diaminoheptaneand with amino acceptors other than 2-oxoglutarate (e.g., pyruvate,2-oxobutanoate) has been reported (Samsonova et al., supra; Kim, K. H.,J Biol Chem 239:783-786 (1964)). A putrescine aminotransfemse withhigher activity with pyruvate as the amino acceptor thanalpha-ketoglutamte is the spuC gene of Pseudomonas aeruginosa (Lu etal., J Bacteriol 184:3765-3773 (2002)).

Gene name GI Number GenBank ID Organism ygjG 145698310 NP_417544Escherichia coli spuC 9946143 AAG03688 Pseudomonas aeruginosa

Yet additional candidate enzymes include beta-alanine/alpha-ketoglutamteaminotransferases which produce malonate semialdehyde from beta-alanine(WO08027742). Exemplary enzymes are shown in the table below.

Gene name GI Number GenBank ID Organism SkyPYD4 98626772 ABF58893.1Saccharomyces kluyveri SkUGA1 98626792 ABF58894.1 Saccharomyces kluyveriUGA1 6321456 NP_011533.1 Saccharomyces cerevisiae Abat 122065191P50554.3 Rattus norvegicus Abat 120968 P80147.2 Sus scrofa

Step G—6-Aminocaproyl-CoA/Acyl-CoA Transferase or 6-Aminocaproyl-CoASynthase.

2.8.3.a Coenzyme-A Transferase.

CoA transferases catalyze reversible reactions that involve the transferof a CoA moiety from one molecule to another. For example, step G can becatalyzed by a 6-aminocaproyl-CoA/Acyl CoA transferase. One candidateenzyme for these steps is the two-unit enzyme encoded by pcaI and pcaJin Pseudomonas, which has been shown to have 3-oxoadipyl-CoA/succinatetransferase activity ((Kaschabek and Reineke, J. Bacteriol. 177:320-325(1995); and Kaschabek. and Reineke, J. Bacteriol. 175:6075-6081 (1993)).Similar enzymes based on homology exist in Acinetobacter sp. ADP1(Kowalchuk et al., Gene 146:23-30 (1994)) and Streptomyces coelicolor.Exemplary enzymes are shown in the table below.

Gene name GI Number GenBank ID Organism pcaI 24985644 AAN69545.1Pseudomonas putida pcaJ 26990657 NP_746082.1 Pseudomonas putida pcaI50084858 YP_046368.1 Acinetobacter sp. ADP1 pcaJ 141776 AAC37147.1Acinetobacter sp. ADP1 pcaI 21224997 NP_630776.1 Streptomyces coelicolorpcaJ 21224996 NP_630775.1 Streptomyces coelicolor HPAG1_0676 108563101YP_627417 Helicobacter pylori HPAG1_0677 108563102 YP_627418Helicobacter pylori ScoA 16080950 NP_391778 Bacillus subtilis ScoB16080949 NP_391777 Bacillus subtilis

A 3-oxoacyl-CoA transferase that can utilize acetate as the CoA acceptoris acetoacetyl-CoA transferase, encoded by the E. coli atoA (alphasubunit) and atoD (beta subunit) genes (Vanderwinkel et al., Biochem.Biophys. Res Commun. 33:902-908 (1968); Korolev et al., ActaCrystallogr. D Biol Crystallogr. 58:2116-2121 (2002)). This enzyme hasalso been shown to transfer the CoA moiety to acetate from a variety ofbranched and linear acyl-CoA substrates, including isobutyrate (Matthieset al., Appl Environ Microbiol 58:1435-1439 (1992)), valerate(Vanderwinkel et al., supra) and butanoate (Vanderwinkel et al., supra).Exemplary enzymes are shown in the table below.

Gene name GI Number GenBank ID Organism atoA 2492994 P76459.1Escherichia coli K12 atoD 2492990 P76458.1 Escherichia coli K12 actA62391407 YP_226809.1 Corynebacterium glutamicum ATCC 13032 cg059262389399 YP_224801.1 Corynebacterium glutamicum ATCC 13032 ctfA 15004866NP_149326.1 Clostridium acetobutylicum ctfB 15004867 NP_149327.1Clostridium acetobutylicum ctfA 31075384 AAP42564.1 Clostridiumsaccharoperbutylacetonicum ctfB 31075385 AAP42565.1 Clostridiumsaccharoperbutylacetonicum

The above enzymes may also exhibit the desired activities on6-aminocaproate and 6-aminocaproyl-CoA, as in step G. Nevertheless,additional exemplary transferase candidates are catalyzed by the geneproducts of cat1, cat2, and cat3 of Clostridium kluyveri which have beenshown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoAtransferase activity, respectively (Seedorf et al., supra; Sohling etal., Eur. J Biochem. 212:121-127 (1993); Sohling et al., J Bacteriol.178:871-880 (1996)).

Gene name GI Number GenBank ID Organism cat1 729048 P38946.1 Clostridiumkluyveri cat2 172046066 P38942.2 Clostridium kluyveri cat3 146349050EDK35586.1 Clostridium kluyveri

The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobicbacterium Acidaminococcus fermentans reacts with diacid glutaconyl-CoAand 3-butenoyl-CoA (Mack et al., FEBS Lett. 405:209-212 (1997)). Thegenes encoding this enzyme are gctA and gctB. This enzyme has reducedbut detectable activity with other CoA derivatives includingglutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckelet al., Eur. J. Biochem. 118:315-321 (1981)). The enzyme has been clonedand expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51(1994)).

Gene name GI Number GenBank ID Organism gctA 559392 CAA57199.1Acidaminococcus fermentans gctB 559393 CAA57200.1 Acidaminococcusfermentans

EC 6.2.1.a Acid-Thiol Ligase.

Step G can also involve an acid-thiol ligase or synthetase functionality(the terms ligase, synthetase, and synthase are used hereininterchangeably and refer to the same enzyme class). Exemplary genesencoding enzymes to may out these transformations include the sucCDgenes of E. coli which naturally form a succinyl-CoA synthetase complex.This enzyme complex naturally catalyzes the formation of succinyl-CoAfrom succinate with the contaminant consumption of one ATP, a reactionwhich is reversible in vivo (Buck et al., Biochem. 24:6245-6252 (1985)).Given the structural similarity between succinate and adipate, that is,both are straight chain dicarboxylic acids, it is reasonable to expectsome activity of the sucCD enzyme on adipyl-CoA.

Gene name GI Number GenBank ID Organism sucC 16128703 NP_415256.1Escherichia coli sucD 1786949 AAC73823.1 Escherichia coli

Additional exemplary CoA-ligases include the rat dicarboxylate-CoAligase for which the sequence is yet uncharacterized (Vamecq et al.,Biochemical Journal 230:683-693 (1985)), either of the two characterizedphenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al.,Biochem. J. 395:147-155 (2005); Wang et al., Biochem Biophy Res Commun360(2):453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonasputida (Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)),and the 6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Boweretal., J. Bacteriol. 178(14):4122-4130 (1996)). Exemplary enzymes areshown in the table below

Gene name GI Number GenBank ID Organism phl 77019264 CAJ15517.1Penicillium chrysogenum phlB 152002983 ABS19624.1 Penicilliumchrysogenum paaF 22711873 AAC24333.2 Pseudomonas putida bioW 50812281NP_390902.2 Bacillus subtilis AACS 21313520 NP_084486.1 Mus musculusAACS 31982927 NP_076417.2 Homo sapiens

ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is anothercandidate enzyme that couples the conversion of acyl-CoA esters to theircorresponding acids with the concurrent synthesis of ATP. Severalenzymes with broad substrate specificities have been described in theliterature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, wasshown to operate on a variety of linear and branched-chain substratesincluding acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate,butyrate, isobutyryate, isovalerate, succinate, fumarate, phenylacetate,indoleacetate (Musfeldt et al., J Bacteriol 184:636-644 (2002)). Theenzyme from Haloarcula marismortui (annotated as a succinyl-CoAsynthetase) accepts propionate, butyrate, and branched-chain acids(isovalerate and isobutyrate) as substrates, and was shown to operate inthe forward and reverse directions (Brasen et al., Arch Microbiol182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophiliccrenarchaeon Pyrobaculum aerophilum showed the broadest substrate rangeof all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA(preferred substrate) and phenylacetyl-CoA (Brasen et al., supra). Theenzymes from A. fulgidus, H marismortui and P. aerophilum have all beencloned, functionally expressed, and characterized in E. coli (Musfeldtet al., supra; Brasen et al., supra).

Gene name GI Number GenBank ID Organism AF1211 11498810 NP_070039.1Archaeoglobus fulgidus DSM 4304 Scs 55377722 YP_135572.1 Haloarculamarismortui ATCC 43049 PAE3250 18313937 NP_560604.1 Pyrobaculumaerophilum str. IM2

Yet another option is to employ a set of enzymes with net ligase orsynthetase activity. For example, phosphotransadipylase and adipatekinase enzymes are catalyzed by the gene products of buk1, buk2, and ptbfrom C. acetobutylicum (Walter et al., Gene 134:107-111 (1993); Huang etal., J. Mol. Microbial. Biotechnol. 2:33-38 (2000)). The ptb geneencodes an enzyme that can convert butyryl-CoA into butyryl-phosphate,which is then converted to butyrate via either of the buk gene productswith the concomitant generation of ATP.

Gene name GI Number GenBank ID Organism ptb 15896327 NP_349676Clostridium acetobutylicum buk1 15896326 NP_349675 Clostridiumacetobutylicum buk2 20137415 Q97II1 Clostridium acetobutylicum

Step H—Amidohydrolase. EC 6.3.1.a/6.3.2.a Amide Synthases/PeptideSynthases.

The direct conversion of 6-aminocaproate to caprolactam as in step H caninvolve the formation of an intramolecular peptide bond. Ribosomes,which assemble amino acids into proteins during translation, arenature's most abundant peptide bond-forming catalysts. Nonribosomalpeptide synthetases are peptide bond forming catalysts that do notinvolve messenger mRNA (Schwarzer et al., Nat Prod. Rep. 20:275-287(2003)). Additional enzymes capable of forming peptide bonds are shownin the table below.

Gene name GI Number GenBank ID Organism acsA 60650089 BAD90933Pseudomonas chlororaphis puuA 87081870 AAC74379 Escherichia coli bls41016784 Q9R8E3 Streptomyces clavuligerus

Step I—Spontaneous Cyclization.

The conversion of 6-aminocaproyl-CoA to caprolactam can occur byspontaneous cyclization. Because 6-aminocaproyl-CoA can cyclizespontaneously to caprolactam, it eliminates the need for a dedicatedenzyme for this step. A similar spontaneous cyclization is observed with4-aminobutyryl-CoA which forms pyrrolidinone (Ohsugi et al., J Biol Chem256:7642-7651 (1981)).

Step J—6-Aminocaproyl-CoA Reductase (Aldehyde Forming).

The transformation of 6-aminocaproyl-CoA to 6-aminocaproate semialdehydeas in step J can involve an acyl-CoA dehydrogenases capable of reducingan acyl-CoA to its corresponding aldehyde. An EC 1.2.1.b oxidoreductase(acyl-CoA to aldehyde) provides suitable enzyme activity. Exemplaryenzymes in this class are described above.

Step K—HMDA Transaminase or HMDA Dehydrogenase.

EC 1.4.1.a Oxidoreductase Operating on Amino Acids.

Step K depicts a reductive animation and entails the conversion of6-aminocaproate semialdehyde to hexamethylenediamine.

Most oxidoreductases operating on amino acids catalyze the oxidativedeamination of alpha-amino acids with NAD+ or NADP+ as acceptor, thoughthe reactions are typically reversible. Exemplary oxidoreductasesoperating on amino acids of the EC 1.4.1. class are described elsewhereherein, for example those for Step F acting on 6-aminocaproatetransaminase or 6-aminocaproate dehydrogenase. The lysine6-dehydrogenase (deaminating), encoded by the lysDH genes, catalyze theoxidative deamination of the epsilon-amino group of L-lysine to form2-aminoadipate-6-semialdehyde, which in turn nonenzymatically cyclizesto form A′-piperideine-6-carboxylate (Misono et al., J. Bacteriol.150:398-401 (1982)). Exemplary enzymes can be found in Geobacillusstearothermophilus (Heydari et al., Appl Environ. Microbiol 70:937-942(2004)), Agrobacterium tumefaciens (Hashimoto et al., J Biochem106:76-80 (1989); Misono et al., supra), and Achromobacter denitrificans(Ruldeekulthamrong et al., BMB. Rep. 41:790-795 (2008)). Such enzymesare particularly good candidates for converting adipate semialdehyde to6-aminocaproate given the structural similarity between adipatesemialdehyde and 2-aminoadipate-6-semialdehyde.

Gene name GI Number GenBank ID Organism lysDH 13429872 BAB39707Geobacillus stearothermophilus lysDH 15888285 NP_353966 Agrobacteriumtumefaciens lysDH 74026644 AAZ94428 Achromobacter denitrificans

EC 2.6.1.a Aminotransferase.

Step K, in certain embodiments, can involve the transamination of a6-aldehyde to an amine. This transformation can be catalyzed bygamma-aminobutyrate transaminase (GABA transaminase). Examples arebelow.

Gene name GI Number GenBank ID Organism gabT 16130576 NP_417148.1Escherichia coli puuE 16129263 NP_415818.1 Escherichia coli abat37202121 NP_766549.2 Mus musculus gabT 70733692 YP_257332.1 Pseudomonasfluorescens abat 47523600 NP_999428.1 Sus scrofa

Additional enzyme candidates include putrescine aminotransferases orother diamine aminotransferases. Such enzymes are particularly wellsuited for carrying out the conversion of 6-aminocaproate semialdehydeto hexamethylenediamine. Examples are described below and elsewhereherein.

Gene name GI Number GenBank ID Organism ygjG 145698310 NP_417544Escherichia coli spuC 9946143 AAG03688 Pseudomonas aeruginosa

Yet additional candidate enzymes includebeta-alanine/alpha-ketoglutarate aminotransferases which producemalonate semialdehyde from beta-alanine (WO08027742). Exemplarycandidates are described elsewhere herein, such as for Step F. StepL—Adipyl-CoA Hydrolase, Adipyl-CoA Ligase, Adipyl-CoA Transferase orPhosphotransadipylase/Adipate Kinase. Step L can involve adipyl-CoAsynthetase (also referred to as adipate-CoA ligase),phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase,or adipyl-CoA hydrolase. From an energetic standpoint, it is desirablefor the final step in the adipate synthesis pathway to be catalyzed byan enzyme or enzyme pair that can conserve the ATP equivalent stored inthe thioester bond of adipyl-CoA. The product of the sucC and sucD genesof E. coli, or homologs thereof, can potentially catalyze the finaltransformation shown in FIG. 8 should they exhibit activity onadipyl-CoA. The sucCD genes naturally form a succinyl-CoA synthetasecomplex that catalyzes the formation of succinyl-CoA from succinate withthe concaminant consumption of one ATP, a reaction which is reversiblein vivo (Buck et al., Biochem. 24:6245-6252 (1985)). Given thestructural similarity between succinate and adipate, that is, both arestraight chain dicarboxylic acids, it is reasonable to expect someactivity of the sucCD enzyme on adipyl-CoA. An enzyme exhibitingadipyl-CoA ligase activity can equivalently carry out the ATP-generatingproduction of adipate from adipyl-CoA, here using AMP and PPi ascofactors, when operating in the opposite physiological. ExemplaryCoA-ligases include the rat dicarboxylate-CoA ligase for which thesequence is yet uncharacterized (Vamecq et al., Biochem. J. 230:683-693(1985)), either of the two characterized phenylacetate-CoA ligases fromP. chrysogenum (Lamas-Maceiras et al., Biochem. J 395, 147-155 (2005);Wang et al., Biochem. Biophy. Res. Commun. 360:453-458 (2007)), thephenylacetate-CoA ligase from Pseudomonas putida (Martinez-Blanco etal., J. Biol. Chem. 265:7084-7090 (1990)), and the6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower et al., J.Bacteriol. 178:4122-4130 (1996)). The protein sequences for each ofthese exemplary gene products can be found using the following GInumbers and/or GenBank identifiers:

Gene name GI Number GenBank ID Organism sucC 16128703 NP_415256.1Escherichia coli sucD 1786949 AAC73823.1 Escherichia coli

Another option, using phosphotransadipylase/adipate kinase, is catalyzedby the gene products of buk1, buk2, and ptb from C. acetobutylicum(Walter et al., Gene 134:107-111 (1993); Huang et al., J. Mol.Microbiol. Biotechnol. 2:33-38 (2000)), or homologs thereof. The ptbgene encodes an enzyme that can convert butyryl-CoA intobutyryl-phosphate, which is then converted to butyrate via either of thebuk gene products with the concomitant generation of ATP. The analogousset of transformations, that is, conversion of adipyl-CoA toadipyl-phosphate followed by conversion of adipyl-phosphate to adipate,can be carried out by the buk1, buk2, and ptb gene products. The proteinsequences for each of these exemplary gene products can be found usingthe following GI numbers and/or GenBank identifiers:

Gene name GI Number GenBank ID Organism ptb 15896327 NP_349676Clostridium acetobutylicum buk1 15896326 NP_349675 Clostridiumacetobutylicum buk2 20137415 Q97II1 Clostridium acetobutylicum

Alternatively, an acetyltransferase capable of transferring the CoAgroup from adipyl-CoA to acetate can be applied. Similar transformationsare catalyzed by the gene products of cat1, cat2, and cat3 ofClostridium kluyveri which have been shown to exhibit succinyl-CoA,4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferase activity,respectively (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996);Seedorf et al., Proc. Natl. Acad. Sci. USA 105:2128-2133 (2008)). Theprotein sequences for each of these exemplary gene products can be foundusing the following GI numbers and/or GenBank identifiers:

Gene name GI Number GenBank ID Organism cat1 729048 P38946.1 Clostridiumkluyveri cat2 172046066 P38942.2 Clostridium kluyveri cat3 146349050EDK35586.1 Clostridium kluyveri

Finally, though not as desirable from an energetic standpoint, theconversion of adipyl-CoA to adipate can also be carried out by anacyl-CoA hydrolase or equivalently a thioesterase. The top E. coli genecandidate is tesB (Naggert et al., J. Biol. Chem. 266:11044-11050(1991)), which shows high similarity to the human acot8, which is adicarboxylic acid acetyltransferase with activity on adipyl-CoA (Westinet al., J. Biol. Chem. 280:38125-38132 (2005)). This activity has alsobeen characterized in the rat liver (Deana, Biochem. Int. 26:767-773(1992)). The protein sequences for each of these exemplary gene productscan be found using the following GI numbers and/or GenBank identifiers:

Gene name GI Number GenBank ID Organism tesB 16128437 NP_414986Escherichia coli acot8 3191970 CAA15502 Homo sapiens acot8 51036669NP_570112 Rattus norvegicus

Other native candidate genes include those in the table below. Theprotein sequences for each of these exemplary gene products can be foundusing the following GI numbers and/or GenBank identifiers:

Gene name GI Number GenBank ID Organism tesA 16128478 NP_415027Escherichia coli ybgC 16128711 NP_415264 Escherichia coli paaI 16129357NP_415914 Escherichia coli ybdB 16128580 NP_415129 Escherichia coli

EC 2.8.3.a Coenzyme-A Transferase.

CoA transferases catalyze reversible reactions that involve the transferof a CoA moiety from one molecule to another. For example, step L can becatalyzed by a adipyl-CoA transferase. One candidate enzyme for thisstep is the two-unit enzyme encoded by pcaI and pcaJ in Pseudomonas,which has been shown to have 3-oxoadipyl-CoA/succinate transferaseactivity (Kaschabek and Reineke, J Bacteriol. 177:320-325 (1995); andKaschabek. and Reineke, J. Bacteriol. 175:6075-6081 (1993)). Similarenzymes based on homology exist in Acinetobacter sp. ADP1 (Kowalchuk etal., Gene 146:23-30 (1994)) and Streptomyces coelicolor. Additionalexemplary succinyl-CoA:3:oxoacid-CoA transferases are present inHelicobacter pylori (Corthesy-Theulaz et al., J. Biol. Chem.272:25659-25667 (1997)) and Bacillus subtilis (Stols et al., Protein.Expr. Punf. 53:396-403 (2007)).

Gene name GI Number GenBank ID Organism pcaI 24985644 AAN69545.1Pseudomonas putida pcaJ 26990657 NP_746082.1 Pseudomonas putida pcaI50084858 YP_046368.1 Acinetobacter sp. ADP1 pcaJ 141776 AAC37147.1Acinetobacter sp. ADP1 pcaI 21224997 NP_630776.1 Streptomyces coelicolorpcaJ 21224996 NP_630775.1 Streptomyces coelicolor HPAG1_0676 108563101YP_627417 Helicobacter pylori HPAG1_0677 108563102 YP_627418Helicobacter pylori ScoA 16080950 NP_391778 Bacillus subtilis ScoB16080949 NP_391777 Bacillus subtilis

A 3-oxoacyl-CoA transferase that can utilize acetate as the CoA acceptoris acetoacetyl-CoA transferase, encoded by the E. coli atoA (alphasubunit) and atoD (beta subunit) genes (Vanderwinkel et al., Biochem.Biophys. Res Commun. 33:902-908 (1968); Korolev et al., ActaCrystallogr. D Biol Crystallogr. 58:2116-2121 (2002)). Exemplarycandidates are shown in the table below.

Gene name GI Number GenBank ID Organism atoA 2492994 P76459.1Escherichia coli K12 atoD 2492990 P76458.1 Escherichia coli K12 actA62391407 YP_226809.1 Corynebacterium glutamicum ATCC 13032 cg059262389399 YP_224801.1 Corynebacterium glutamicum ATCC 13032 ctfA 15004866NP_149326.1 Clostridium acetobutylicum ctfB 15004867 NP_149327.1Clostridium acetobutylicum ctfA 31075384 AAP42564.1 Clostridiumsaccharoperbutylacetonicum ctfB 31075385 AAP42565.1 Clostridiumsaccharoperbutylacetonicum

The above enzymes may also exhibit the desired activities on adipyl-CoAand adipate for step L. Nevertheless, additional exemplary transferasecandidates are catalyzed by the gene products of cat1, cat2, and cat3 ofClostridium kluyveri which have been shown to exhibit succinyl-CoA,4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity, respectively(Seedorf et al., Proc. Natl. Acad. Sci U S. A 105:2128-2133 (2008);Sohling et al., Eur. J Biochem. 212:121-127 (1993); Sohling et al., JBacterial. 178:871-880 (1996)).

Gene name GI Number GenBank ID Organism cat1 729048 P38946.1 Clostridiumkluyveri cat2 172046066 P38942.2 Clostridium kluyveri cat3 146349050EDK35586.1 Clostridium kluyveri

The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobicbacterium Acidaminococcus fermentans reacts with diacid glutaconyl-CoAand 3-butenoyl-CoA (Mack et al., FEBS Lett. 405:209-212 (1997)). Thegenes encoding this enzyme are gctA and gctB. This enzyme has reducedbut detectable activity with other CoA derivatives includingglutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckelet al., Eur. J. Biochem. 118:315-321 (1981)). The enzyme has been clonedand expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51(1994)).

Gene name GI Number GenBank ID Organism gctA 559392 CAA57199.1Acidaminococcus fermentans gctB 559393 CAA57200.1 Acidaminococcusfermentans

Example X Methacrylic Acid Synthesis Enzymes

This Example provides genes that can be used for conversion ofsuccinyl-CoA to methacrylic acid as depicted in the pathways of FIG. 9.

FIG. 9. depicts 3-Hydroxyisobutymte and methacrylic acid production arecarried out by the following enzymes: A) Methylmalonyl-CoA mutase, B)Methylmalonyl-CoA epimerase, C) Methylmalonyl-CoA reductase (aldehydelimning), D) Methylmalonate semialdehyde reductase, E)3-hydroxyisobutyrate dehydratase, F) Methylmalonyl-CoA reductase(alcohol forming).

Step A—Methylmalonyl-CoA mutase (designated as EMA2).

Methylmalonyl-CoA mutase (MCM) (EMA2) (EC 5.4.99.2) is acobalamin-dependent enzyme that converts succinyl-CoA tomethylmalonyl-CoA. In E coli, the reversible adenosylcobalamin-dependentmutase participates in a three-step pathway leading to the conversion ofsuccinate to propionate (Haller et al., Biochemistry 39:4622-4629(2000)). Overexpression of the EMA2 gene candidate along with thedeletion of YgfG can be used to prevent the decarboxylation ofmethylmalonyl-CoA to propionyl-CoA and to maximize the methylmalonyl-CoAavailable for MAA synthesis. EMA2 is encoded by genes scpA inEscherichia coli (Bobik and Rasche, Anal. Bioanal. Chem. 375:344-349(2003); Haller et al., Biochemistry 39:4622-4629 (2000)) and mutA inHomo sapiens (Padovani and Banenee, Biochemistry 45:9300-9306 (2006)).In several other organisms EMA2 contains alpha and beta subunits and isencoded by two genes. Exemplary gene candidates encoding the two-subunitprotein are Propionibacterium fredenreichii sp. shermani mutA and mutB(Korotkova and Lidstrom, J Biol. Chem. 279:13652-13658 (2004)),Methylobacterium extorquens mcmA and mcmB (Korotkova and Lidstrom,supra, 2004), and Ralstonia eutropha sbm1 and sbm2 (Peplinski et al.,Appl. Microbiol. Biotech. 88:1145-59 (2010)). Additional enzymecandidates identified based on high homology to the E. coli spcA geneproduct are also listed below.

Protein GenBank ID GI Number Organism scpA NP_417392.1 16130818Escherichia coli K12 mutA P22033.3 67469281 Homo sapiens mutA P11652.3127549 Propionibacterium fredenreichii sp. shermanii mutB P11653.3127550 Propionibacterium fredenreichii sp. shermanii mcmA Q84FZ175486201 Methylobacterium extorquens mcmB Q6TMA2 75493131Methylobacterium extorquens Sbm1 YP_724799.1 113866310 Ralstoniaeutropha H16 Sbm2 YP_726418.1 113867929 Ralstonia eutropha H16 sbmNP_838397.1 30064226 Shigella flexneri SARI_04585 ABX24358.1 160867735Salmonella enterica YfreA_01000861 ZP_00830776.1 77975240 Yersiniafrederiksenii

These sequences can be used to identify homologue proteins in GenBank orother databases through sequence similarity searches (for example,BLASTp). The resulting homologue proteins and their corresponding genesequences provide additional exogenous DNA sequences for transformationinto E. coli or other suitable host microorganisms to generateproduction hosts. Additional gene candidates include the following,which were identified based on high homology to the E. coli spcA geneproduct.

There further exists evidence that genes adjacent to the EMA2 catalyticgenes contribute to maximum activity. For example, it has beendemonstrated that the meaB gene from M. extorquens forms a complex withEMA2, stimulates in vitro mutase activity, and possibly protects it fromirreversible inactivation (Korotkova and Lidstrom, J. Biol. Chem.279:13652-13658 (2004)). The M. extorquens meaB gene product is highlysimilar to the product of the E coli argKgene (BLASTp: 45% identity,e-value: 4e-67), which is adjacent to scpA on the chromosome. Nosequence for a meaB homolog in P. freudenreichii is catalogued inGenBank. However, the Propionibacterium acnes KPA171202 gene product, YP055310.1, is 51% identical to the M. extorquens meaB protein and itsgene is also adjacent to the EMA2 gene on the chromosome. A similar geneis encoded by H16_B1839 of Ralstonia eutropha H16.

Gene GenBank ID GI Number Organism argK AAC75955.1 1789285 Escherichiacoli K12 PPA0597 YP_055310.1 50842083 Propionibacterium acnes KPA1712022QM8_B 158430328 Methylobacterium extorquens H16_B1839 YP_841351.1116695775 Ralstonia eutropha H16

E. coli can synthesize adenosylcobalamin, a necessary cofactor for thisreaction, only when supplied with the intermediates cobinamide orcobalamin (Lawrence and Roth. J. Bacteriol. 177:6371-6380 (1995);Lawrence and Roth, Genetics 142:11-24 (1996)). Alternatively, theability to synthesize cobalamins de novo has been conferred upon E colifollowing the expression of heterologous genes (Raux et al., J.Bacteriol. 178:753-767 (1996)).

Alternatively, isobutyryl-CoA mutase (ICM) (EC 5.4.99.13) could catalyzethe proposed transformation shown in FIG., step B. ICM is acobalamin-dependent methylmutase in the EMA2 family that reversiblyrearranges the carbon backbone of butyryl-CoA into isobutyryl-CoA(Ratnatilleke et al., J. Biol. Chem. 274:31679-31685 (1999)). A recentstudy of a novel ICM in Methylibium petroleiphilum, along with previouswork, provides evidence that changing a single amino acid near theactive site alters the substrate specificity of the enzyme (Ratnatillekeet al., J. Biol. Chem. 274:31679-31685 (1999); Rohwerder et al., Appl.Environ. Microbiol. 72:4128-4135. (2006)). This indicates that, if anative enzyme is unable to catalyze or exhibits low activity for theconversion of 4HB-CoA to 3HIB-CoA, the enzyme can be rationallyengineered to increase this activity. Exemplary genes/proteins areidentified below.

Gene GenBank ID GI Number Organism icmA CAB40912.1 4585853 Streptomycescoelicolor A3(2) Mpe B0541 YP_001023546.1 124263076 Methylibiumpetroleiphilum PM1 icm AAC08713.1 3002492 Streptomyces cinnamonensisicmB CAB59633.1 6137077 Streptomyces cinnamonensis icmA NP_824008.129829374 Streptomyces avermitilis icmB NP_824637.1 29830003 Streptomycesavermitilis

Step B—Methylmalonyl-CoA epimerase (designated as EMA3).

Methylmalonyl-CoA epimerase (MMCE) (EMA3) is the enzyme thatinterconverts (R)-methylmalonyl-CoA and (S)-methylmalonyl-CoA. EMA3 isan essential enzyme in the breakdown of odd-numbered fatty acids and ofthe amino acids valine, isoleucine, and methionine. EMA3 activity is notbelieved to be encoded in the E. coli genome (Boynton et al., J.Bacteriol. 178:3015-3024 (1996)), but is present in other organisms;gene candidates include those shown below. This enzymatic step may ormay not be necessary depending upon the stereospecificity of the enzymeor enzymes used for the conversion of methylmalonyl-CoA to 3-HIB. Thesegenes/proteins are described below.

Gene GenBank ID GI Number Organism YqjC NP_390273 255767522 Bacillussubtilis MCEE Q96PE7.1 50401130 Homo sapiens Mcee_predictedNP_001099811.1 157821869 Rattus norvegicus AF454511 AAL57846.1 18042135Propionibacterium fredenreichii sp. shermanii Mmce AAT92095.1 51011368Caenorhabditis elegans AE016877 AAP08811.1 29895524 Bacillus cereus ATCC14579

Step C—Methylmalonyl-CoA reductase (aldehyde forming) (designated asEMA4).

The reduction of methylmalonyl-CoA to its corresponding aldehyde,methylmalonate semialdehyde, is catalyzed by a CoA-dependent aldehydedehydrogenase (EC 1.2.1.-). Conversion of methylmalonyl-CoA tomethylmalonic semialdehyde, is accomplished by a CoA-dependent aldehydedehydrogenase. An enzyme encoded by a malonyl-CoA reductase gene fromSulfolobus tokodaii (Alber et. al., J. Bacteriol 188(24):8551-8559(2006)), has been shown to catalyze the conversion of methylmalonyl-CoAto its corresponding aldehyde (WO2007141208). A similar enzyme exists inMetallosphaera sedula (Alber et. al., J. Bacteriol. 188(24):8551-8559(2006)). Several additional CoA dehydrogenases are capable also ofreducing an acyl-CoA to its corresponding aldehyde. The reduction ofmethylmalonyl-CoA to its corresponding aldehyde, methylmalonatesemialdehyde, is catalyzed by a CoA-dependent aldehyde dehydrogenase.Exemplary enzymes include fatty acyl-CoA reductase, succinyl-CoAreductase (EC 1.2.1.76), acetyl-CoA reductase and butyryl-CoA reductase.Exemplary fatty acyl-CoA reductase enzymes are shown below. Also knownis a CoA- and NADP-dependent succinate semialdehyde dehydrogenase (alsoreferred to as succinyl-CoA reductase) encoded by the sucD gene inClostridium kluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880(1996); Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)) andsucD of P. gingivalis (Takahashi, J. Bacteriol 182:4704-4710 (2000)).Additional succinyl-CoA reductase enzymes participate in the3-hydroxypropionate/4-hydroxybutyrate cycle of thermophilic archaea.Exemplary enzymes are shown in the following table.

Protein GenBank ID GI Number Organism acr1 YP_047869.1 50086359Acinetobacter calcoaceticus acr1 AAC45217 1684886 Acinetobacter baylyiacr1 BAB85476.1 18857901 Acinetobacter sp. Strain M-1 MSED_0709YP_001190808.1 146303492 Metallosphaera sedula Tneu_0421 Thermoproteusneutrophilus sucD P38947.1 172046062 Clostridium kluyveri sucDNP_904963.1 34540484 Porphyromonas gingivalis bphG BAA03892.1 425213Pseudomonas sp adhE AAV66076.1 55818563 Leuconostoc mesenteroides bldAAP42563.1 31075383 Clostridium saccharoperbutylacetonicum

An additional enzyme type that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase which transforms malonyl-CoA tomalonic semialdehyde. Malonyl-CoA reductase is a key enzyme inautotrophic carbon fixation via the 3-hydroxypropionate cycle inthemoacidophilic archaeal bacteria (Berg, Science 318:1782-1786 (2007);and Thauer, Science 318:1732-1733 (2007)). The enzyme utilizes NADPH asa cofactor and has been characterized in Metallosphaera and Sulfolobussp. (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Hugler, J.Bacteriol. 184:2404-2410 (2002)). Exemplary enzymes include those in thefollowing table.

Gene GenBank ID GI Number Organism Msed 0709 YP_001190808.1 146303492Metallosphaera sedula Mcr NP_378167.1 15922498 Sulfolobus tokodaii asd-2NP_343563.1 15898958 Sulfolobus solfataricus Saci 2370 YP_256941.170608071 Sulfolobus acidocaldarius Ald AAT66436 49473535 Clostridiumbeijerinckii eutE AAA80209 687645 Salmonella typhimurium eutE P774452498347 Escherichia coli

A bifunctional enzyme with acyl-CoA reductase and alcohol dehydrogenaseactivity can directly convert methylmalonyl-CoA to 3-HIB. Exemplarybifunctional oxidoreductases that convert an acyl-CoA to alcohol includethose that transform substrates such as acetyl-CoA to ethanol (forexample, aahE from E. coli (Kessler et al., FEBS Lett. 281:59-63(1991))) and butyryl-CoA to butanol (for example, adhE2 from C.acetobutylicum (Fontaine et al., J. Bacteriol. 184:821-830 (2002)).Exemplary enzymes are those in the table below.

Protein GenBank ID GI Number Organism adhE NP_415757.1 16129202Escherichia coli adhE2 AAK09379.1 12958626 Clostridium acetobutylicumadhE AAV66076.1 55818563 Leuconostoc mesenteroides bdh I NP_349892.115896543 Clostridium acetobutylicum bdh II NP_349891.1 15896542Clostridium acetobutylicum Mcr AAS20429.1 42561982 Chloroflexusaurantiacus Rcas 2929 YP_001433009.1 156742880 Roseiflexus castenholziiNAP1_02720 ZP_01039179.1 85708113 Erythrobacter sp. NAP1 MGP2080_00535ZP_01626393.1 119504313 marine gamma proteobacterium HTCC2080

Step D—Methylmalonate Semialdehyde Reductase (Designated as EMA5).

The reduction of methylmalonate semialdehyde to is catalyzed by EMA5 or3-HIB dehydrogenase. This enzyme participates in valine, leucine andisoleucine degradation and has been identified in bacteria, eukaryotes,and mammals. Exemplary enzymes are shown below.

Protein GenBank ID GI Number Organism P84067 P84067 75345323 Thermusthermophilus 3hidh P31937.2 12643395 Homo sapiens 3hidh P32185.1 416872Oryctolagus cuniculus mmsB NP_746775.1 26991350 Pseudomonas putida mmsBP28811.1 127211 Pseudomonas aeruginosa dhat Q59477.1 2842618 Pseudomonasputida

Step E—3-HIB Dehydratase (designated as EMA6).

The dehydration of 3-HIB to MAA is catalyzed by an enzyme with EMA6activity (EC 4.2.1.-). The final step involves the dehydration of 3-HIBto MAA The dehydration of 3-HIB to MAA is catalyzed by an enzyme withEMA6 activity. Although no direct evidence for this specific enzymatictransformation has been identified, most dehydratases catalyze thealpha,beta-elimination of water, which involves activation of the□-hydrogen by an electron-withdrawing carbonyl, carboxylate, orCoA-thiol ester group and removal of the hydroxyl group from thebeta-position (Buckel and Barker, J Bacteriol. 117:1248-1260 (1974);Martins et al, Proc. Natl. Acad Sci. USA 101:15645-15649 (2004)). Thisis the exact type of transformation proposed for the final step in themethacrylate pathway. In addition, the proposed transformation is highlysimilar to the 2-(hydroxymethyl)glutamte dehydratase of Eubacteriumbarkeri, which can catalyze the conversion of 2-hydroxymethyl glutarateto 2-methylene glutarate. This enzyme has been studied in the context ofnicotinate catabolism and is encoded by hmd (Alhapel et al., Proc. Natl.Acad. Sci. USA 103:12341-12346 (2006)). Similar enzymes with highsequence homology are found in Bacteroides capillosus, Anaerotruncuscolihominis, and Natranaerobius thermophilius. Several enzymes are knownto catalyze the alpha, beta elimination of hydroxyl groups. Exemplaryenzymes include 2-(hydroxymethyl)glutarate dehydratase (EC 4.2.1.-),fumarase (EC 4.2.1.2), 2-keto-4-pentenoate dehydratase (EC 4.2.1.80),citramalate hydrolyase and dimethylmaleate hydratase.

2-(Hydroxymethyl)glutarate dehydratase is a [4Fe-4S]-containing enzymethat dehydrates 2-(hydroxymethyl)glutarate to 2-methylene-glutarate,studied for its role in nicontinate catabolism in Eubacteriumbarker/(formerly Clostridium barkeri) (Alhapel et al., Proc Natl AcadSci USA 103:12341-12346 (2006)). Similar enzymes with high sequencehomology are found in Bacteroides capillosus, Anaerotruncus colihominis,and Natranaerobius thermophilius. These enzymes are also homologous tothe alpha- and beta-subunits of [4Fe-4S]-containing bacterial serinedehydratases, for example, E. coli enzymes encoded by tdcG, sdhB, andsdaA). An enzyme with similar functionality in E. barkeri isdimethylmaleate hydratase, a reversible Fe2+-dependent andoxygen-sensitive enzyme in the aconitase family that hydratesdimethylmaeate to form (2R,3S)-2,3-dimethylmalate. This enzyme isencoded by dmdAB (Alhapel et al., Proc Natl Acad Sci USA 103:12341-6(2006); Kollmann-Koch et al., Hoppe Seylers.ZPhysiol Chem. 365:847-857(1984)).

Protein GenBank ID GI Number Organism Hmd ABC88407.1 86278275Eubacterium barkeri BACCAP_02294 ZP_02036683.1 154498305 Bacteroidescapillosus ANACOL_02527 ZP_02443222.1 167771169 Anaerotruncuscolihominis NtherDRAFT_2368 ZP_02852366.1 169192667 Natranaerobiusthermophilus dmdA ABC88408 86278276 Eubacterium barkeri dmdB ABC8840986278277 Eubacterium barkeri

Fumarate hydratase enzymes, which naturally catalyze the reversiblehydration of fumarate to malate. Although the ability of fumaratehydratase to react on branched substrates with 3-oxobutanol as asubstrate has not been described, a wealth of structural information isavailable for this enzyme and other researchers have successfullyengineered the enzyme to alter activity, inhibition and localization(Weaver, Acta Crystallogr. D Biol. Crystallogr. 61:1395-1401 (2005)). E.coli has three fumarases: FumA, FumB, and FumC that are regulated bygrowth conditions. FumB is oxygen sensitive and only active underanaerobic conditions. FumA is active under microanaerobic conditions,and FumC is the only active enzyme in aerobic growth (Tseng et al., J.Bacteriol. 183:461-467 (2001); Woods et al., Biochem. Biophys. Acta954:14-26 (1988); Guest et al., J Gen Microbiol 131:2971-2984 (1985)).Exemplary enzyme candidates include those encoded by fumC fromEscherichia coli and include those in the table below.

Protein GenBank ID GI Number Organism fumA NP_416129.1 16129570Escherichia coli fumB NP_418546.1 16131948 Escherichia coli fumCNP_416128.1 16129569 Escherichia coli fumC O69294 9789756 Campylobacterjejuni fumC P84127 75427690 Thermus thermophilus fumH P14408 120605Rattus norvegicus fuml P93033 39931311 Arabidopsis thaliana fumC Q8NRN839931596 Corynebacterium glutamicum MmcB YP_001211906 147677691Pelotomaculum thermopropionicum MmcC YP_001211907 147677692Pelotomaculum thermopropionicum

Dehydration of 4-hydroxy-2-oxovalerate to 2-oxopentenoate is catalyzedby 4-hydroxy-2-oxovalerate hydratase (EC 4.2.1.80). This enzymeparticipates in aromatic degradation pathways and is typicallyco-transcribed with a gene encoding an enzyme with4-hydroxy-2-oxovalerate aldolase activity. Exemplary gene productsinclude those in the table below. Sequence comparison reveals homologsin a wide range of bacteria, plants and animals. Enzymes with highlysimilar sequences are contained in Klebsiella pneumonia (91% identity,eval=2e-138) and Salmonella enterica (91% identity, eval=4e-138), amongothers.

GenBank Gene Accession No. GI No. Organism mhpD AAC73453.2 87081722Escherichia coli cmtF AAB62293.1 1263188 Pseudomonas putida todGAAA61942.1 485738 Pseudomonas putida cnbE YP_001967714.1 190572008Comamonas sp. CNB-1 mhpD Q13VU0 123358582 Burkholderia xenovorans hpcGCAA57202.1 556840 Escherichia coli C hpaH CAA86044.1 757830 Escherichiacoli W hpaH ABR80130.1 150958100 Klebsiella pneumoniae Sari 01896ABX21779.1 160865156 Salmonella enterica

Another enzyme candidate is citramalate hydrolyase (EC 4.2.1.34), anenzyme that naturally dehydrates 2-methylmalate to mesaconate. Thisenzyme has been studied in Methanocaldococcus jannaschii in the contextof the pyruvate pathway to 2-oxobutanoate, where it has been shown tohave a broad substrate specificity (Drevland et al., J Bacteriol.189:4391-4400 (2007)). This enzyme activity was also detected inClostridium tetanomorphum, Morganella morganii, Citrobacter amalonaticuswhere it is thought to participate in glutamate degradation (Kato etal., Arch. Microbiol 168:457-463 (1997)). The M. jannaschii proteinsequence does not bear significant homology to genes in these organisms.

Protein GenBank ID GI Number Organism leuD Q58673.1 3122345Methanocaldococcus jannaschii

Dimethylmaleate hydratase (EC 4.2.1.85) is a reversible Fe²⁺-dependentand oxygen-sensitive enzyme in the aconitase family that hydratesdimethylmaeate to form (2R,3S)-2,3-dimethylmalate. This enzyme isencoded by dmdAB in Eubacterium barkeri (Alhapel et al., supra;Kollmann-Koch et al., Hoppe Seylers.ZPhysiol Chem. 365:847-857 (1984)).

Protein GenBank ID GI Number Organism dmdA ABC88408 86278276 Eubacteriumbarkeri dmdB ABC88409.1 86278277 Eubacterium barkeri

Step F—Methylmalonyl-CoA Reductase (Alcohol Forming) (Designated asEMA7).

Step F can involve a combined Alcohol/Aldehyde dehydrogenase (EC1.2.1.-). Methylmalonyl-CoA can be reduced to 3-HIB in one step by amultifunctional enzyme with dual acyl-CoA reductase and alcoholdehydrogenase activity. Although the direct conversion ofmethylmalonyl-CoA to 3-HIB has not been reported, this reaction issimilar to the common conversions such as acetyl-CoA to ethanol andbutyryl-CoA to butanol, which are catalyzed by CoA-dependent enzymeswith both alcohol and aldehyde dehydrogenase activities. Gene candidatesinclude the E. coli adhE and exemplary enzymes in the table below.

Protein GenBank ID GI Number Organism Mcr YP_001636209.1 163848165Chloroflexus aurantiacus adhE NP_415757.1 16129202 Escherichia coli bdhINP_349892.1 15896543 Clostridium acetobutylicum bdhII NP_349891.115896542 Clostridium acetobutylicum adhE AAV66076.1 55818563 Leuconostocmesenteroides

Example XI Methacrylic Acid and 2-Hydroxyisobutyric Synthesis Enzymes

This Example provides genes that can be used for conversion ofacetyl-CoA to methacrylic acid and 2-hydroxyisobutyric as depicted inthe pathways of FIG. 10.

FIG. 10. Exemplary pathways enabling production of 2-hydroxyisobutyrateand methacrylic acid from acetyl-CoA. 2-Hydroxyisobutyrate andmethacrylic acid production are carried out by the following enzymes: A)acetyl-CoA:acetyl-CoA acyltransferase, B) acetoacetyl-CoA reductase(ketone reducing), C) 3-hydroxybutyrl-CoA mutase, D)2-hydroxyisobutyryl-CoA dehydratase, E) methacrylyl-CoA synthetase,hydrolase, or transferase, F) 2-hydroxyisobutyryl-CoA synthetase,hydrolase, or transferase.

MAA biosynthesis can proceed from acetyl-CoA in a minimum of fiveenzymatic steps (see FIG. 10). In this pathway, two molecules ofacetyl-CoA are combined to form acetoacetyl-CoA, which is then reducedto 3-hydroxybutyryl-CoA. Alternatively, 4-hydroxybutyryl-CoA can beconverted to 3-hydroxybutyryl-CoA by way of 4-hydroxybutyryl-CoAdehydratase and crotonase (Martins et al., Proc. Nat. Acad. Sci. USA101(44) 15645-15649 (2004); Jones and Woods, Microbiol. Rev. 50:484-524(1986); Berg et al., Science 318(5857) 1782-1786 (2007)). A methylmutasethen rearranges the carbon backbone of 3-hydroxybutyryl-CoA to2-hydroxyisobutyryl-CoA, which is then dehydrated to formmethacrylyl-CoA. Alternatively, 2-hydroxyisobutyryl-CoA can be convertedto 2-hydroxyisobutyrate, secreted, and recovered as product. The finalstep converting methacrylyl-CoA to MAA can be performed by a singleenzyme shown in the figure or a series of enzymes.

A) Acetyl-CoA:Acetyl-CoA Acyltransferase.

Step A involves acetoacetyl-CoA thiolase (EC 2.3.1.9). The formation ofacetoacetyl-CoA from two acetyl-CoA units is catalyzed by acetyl-CoAthiolase. This enzyme is native to E. coli, encoded by gene atoB, andtypically operates in the acetoacetate-degrading direction during fattyacid oxidation (Duncombe and Freman, Arch. Biochem. Biophys. 176:159-170(1976); Freman and Duncombe, Biochim. Biophys. Acta 580:289-297 (1979)).Additional exemplary genes include those below.

Protein GenBank ID GI Number Organism atoB NP_416728 16130161Escherichia coli thlA NP_349476.1 15896127 Clostridium acetobutylicumthlB NP_149242.1 15004782 Clostridium acetobutylicum thl ABA18857.175315385 Clostridium pasteurianum ERG10 NP_015297 6325229 Saccharomycescerevisiae

B) Acetoacetyl-CoA Reductase (Ketone Reducing).

Step B involves acetoacetyl-CoA reductase (EC #: 1.1.1.35). This secondstep entails the reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA byacetoacetyl-CoA reductase. This enzyme participates in the acetyl-CoAfermentation pathway to butyrate in several species of Clostridia andhas been studied in detail (Jones and Woods, Microbiol. Rev. 50:484-524(1986)). The enzyme from Clostridium acetobutylicum, encoded by hbd, hasbeen cloned and functionally expressed in E. coli (Youngleson et al., J.Bacteriol. 171:6800-6807 (1989)). Additionally, subunits of two fattyacid oxidation complexes in E. coli, encoded by fadB and fadJ, functionas 3-hydroxyacyl-CoA dehydrogenases (Binstock and Schulz, MethodsEnzymol. 71 Pt C:403-411 (1981)). Exemplary enzymes are in the followingtable.

Protein GENBANK ID GI NUMBER ORGANISM fadB P21177.2 119811 Escherichiacoli fadJ P77399.1 3334437 Escherichia coli Hbd2 EDK34807.1 146348271Clostridium kluyveri Hbd1 EDK32512.1 146345976 Clostridium kluyveriHSD17B10 O02691.3 3183024 Bos taurus phbB P23238.1 130017 Zoogloearamigera phaB YP_353825.1 77464321 Rhodobacter sphaeroides phaB BAA08358675524 Paracoccus denitrificans Hbd NP_349314.1 15895965 Clostridiumacetobutylicum Hbd AAM14586.1 20162442 Clostridium beijerinckiiMsed_1423 YP_001191505 146304189 Metallosphaera sedula Msed_0399YP_001190500 146303184 Metallosphaera sedula Msed_0389 YP_001190490146303174 Metallosphaera sedula Msed_1993 YP_001192057 146304741Metallosphaera sedula Fox2 Q02207 399508 Candida tropicalis

C) 3-Hydroxybutyrl-CoA Mutase.

Step C involves 3-hydroxybutyryl-CoA mutase (EC 5.4.99.-). In this step,3-hydroxybutyryl-CoA is rearranged to form 2-hydroxyisobutyryl-CoA(2-HIBCoA) by 3-hydroxybutyryl-CoA mutase. This enzyme is a novelICM-like methylmutase recently discovered and characterized inMethylibium petroleiphilum (Ratnatilleke et al., J. Biol. Chem.274:31679-31685 (1999); Rohwerder et al., Appl. Environ. Microbiol.72:4128-4135 (2006)). This enzyme, encoded by Mpe B0541 in Methylibiumpetroleiphilum PM1, has high sequence homology to the large subunit ofmethylmalonyl-CoA mutase in other organisms including Rsph17029_3657 inRhodobacter sphaeroides and Xaut_5021 in Xanthobacter autotrophicus.Changes to a single amino acid near the active site alters the substratespecificity of the enzyme (Ratnatilleke et al., supra, 1999; Rohwerderet al., supra, 2006), so directed engineering of similar enzymes at thissite, such as methylmalonyl-CoA mutase or isobutryryl-CoA mutasedescribed previously, can be used to achieve the desired reactivity.

Gene GenBank ID GI Number Organism Mpe B0541 YP_001023546.1 124263076Methylibium petroleiphilum PM1 Rsphl7029_3657 YP_001045519.1 126464406Rhodobacter sphaeroides Xaut_5021 YP_001409455.1 154243882 Xanthobacterautotrophicus Py2

D) 2-Hydroxyisobutyryl-CoA Dehydratase.

Step D involves 2-hydroxyisobutyryl-CoA dehydratase. The dehydration of2-hydroxyacyl-CoA such as 2-hydroxyisobutyryl-CoA can be catalyzed by aspecial class of oxygen-sensitive enzymes that dehydrate2-hydroxyacyl-CoA derivatives via a radical-mechanism (Buckel andGolding, Annu. Rev. Microbiol. 60:27-49 (2006); Buckel et al., Curr.Opin. Chem. Biol. 8:462-467 (2004); Buckel et al., Biol. Chem.386:951-959 (2005); Kim et al., FEBS 272:550-561 (2005); Kim et al.,FEMS Microbiol. Rev. 28:455-468 (2004); Zhang et al., Microbiology 145(Pt 9):2323-2334 (1999)). Exemplary enzymes are the following.

GenBank Gene Accession No. GI No. Organism hgdA P11569 296439332Acidaminococcus fermentans hgdB P11570 296439333 Acidaminococcusfermentans hgdC P11568 2506909 Acidaminococcus fermentans hadBYP_001086863 126697966 Clostridium difficile hadC YP_001086864 126697967Clostridium difficile hadI YP_001086862 126697965 Clostridium difficilelcdB AJ276553 7242547 Clostridium propionicum

E) Methacrylyl-CoA Synthetase, Hydrolase, or Transferase, and F)2-Hydroxyisobutyryl-CoA Synthetase, Hydrolase, or Transferase.

Steps E and F involve a transferase (EC 2.8.3.-), hydrolase (EC3.1.2.-), or synthetase (EC 6.2.1.-) with activity on a methacrylic acidor 2-hydroxyisobutyric acid, respectively. Direct conversion ofmethacrylyl-CoA to MAA or 2-hydroxyisobutyryl-CoA to2-hydrioxyisobutyrate can be accomplished by a CoA transferase,synthetase or hydrolase. Pathway energetics are most favorable if a CoAtransferase or a CoA synthetase is employed to accomplish thistransformation. In the transferase family, the enzymeacyl-CoA:acetate-CoA transferase, also known as acetate-CoA transferase,is a suitable candidate to catalyze the desired 2-hydroxyisobutyryl-CoAor methacryl-CoA transferase activity due to its broad substratespecificity that includes branched acyl-CoA substrates (Matthies andSchink, Appl. Environ. Microbiol. 58:1435-1439 (1992); Vanderwinkel etal., Biochem. Biophys. Res. Commun. 33:902-908 (1968)). ADP-formingacetyl-CoA synthetase (ACD) is a promising enzyme in the CoA synthetasefamily operating on structurally similar branched chain compounds(Brasen and Schonheit, Arch. Microbiol. 182:277-287 (2004); Musfeldt andSchonheit, J. Bacteriol. 184:636-644 (2002)). In the CoA-hydrolasefamily, the enzyme 3-hydroxyisobutyryl-CoA hydrolase has been shown tooperate on a variety of branched chain acyl-CoA substrates including3-hydroxyisobutyryl-CoA, methylmalonyl-CoA, and3-hydroxy-2-methylbutanoyl-CoA (Hawes et al., Methods Enzymol.324:218-228 (2000); Hawes et al., J. Biol. Chem. 271:26430-26434 (1996);Shimomura et al., J. Biol. Chem. 269:14248-14253 (1994)). Additionalexemplary gene candidates for CoA transferases, synthetases, andhydrolases are discussed elsewhere above.

Example XII Attenuation or Disruption of Endogenous Enzymes

This example provides endogenous enzyme targets for attenuation ordisruption that can be used for enhancing carbon flux through methanoldehydrogenase and formaldehyde assimilation pathways.

DHA Kinase

Methylotrophic yeasts typically utilize a cytosolic DHA kinase tocatalyze the ATP-dependent activation of DHA to DHAP. DHAP together withG3P is combined to form fructose-1,6-bisphosphate (FBP) by FBP aldolase.FBP is then hydrolyzed to F6P by fructose bisphosphatase. The netconversion of DHA and G3P to F6P by this route is energetically costly(1 ATP) in comparison to the F6P aldolase route, described above andshown in FIG. 1. DHA kinase also competes with F6P aldolase for the DHAsubstrate. Attenuation of endogenous DHA kinase activity will thusimprove the energetics of formaldehyde assimilation pathways, and alsoincrease the intracellular availability of DHA for DHA synthase. DHAkinases of Saccharomyces cerevisiae, encoded by DAK1 and DAK2, enablethe organism to maintain low intracellular levels of DHA (Molin et al, JBiol Chem 278:1415-23 (2003)). In methylotrophic yeasts DHA kinase isessential for growth on methanol (Luers et al, Yeast 14:759-71 (1998)).The DHA kinase enzymes of Hansenula polymorpha and Pichia pastoris areencoded by DAK (van der Klei et al, Curr Genet 34:1-11 (1998); Luers etal, supra). DAK enzymes in other organisms can be identified by sequencesimilarity to known enzymes.

Protein GenBank ID GI Number Organism DAK1 NP_013641.1 6323570Saccharomyces cerevisiae DAK2 NP_116602.1 14318466 Saccharomycescerevisiae DAK AAC27705.1 3171001 Hansenula polymorpha DAK AAC39490.13287486 Pichia pastoris DAK2 XP_505199.1 50555582 Yarrowia lipolytica

Methanol Oxidase

Attenuation of redox-inefficient endogenous methanol oxidizing enzymes,combined with increased expression of a cytosolic NADH-dependentmethanol dehydrogenase, will enable redox-efficient oxidation ofmethanol to formaldehyde in the cytosol. Methanol oxidase, also calledalcohol oxidase (EC 1.1.3.13), catalyzes the oxygen-dependent oxidationof methanol to formaldehyde and hydrogen peroxide. In eukaryoticorganisms, alcohol oxidase is localized in the peroxisome. Exemplarymethanol oxidase enzymes are encoded by AOD of Candida boidinii (Sakaiand Tani, Gene 114:67-73 (1992)); and AOX of H. polymorpha, P.methanolica and P. pastoris (Ledeboer et al, Nucl Ac Res 13:3063-82(1985); Koutz et al, Yeast 5:167-77 (1989); Nakagawa et al, Yeast15:1223-1230 (1999)).

Protein GenBank ID GI Number Organism AOX2 AAF02495.1 6049184 Pichiamethanolica AOX1 AAF02494.1 6049182 Pichia methanolica AOX1 AAB57849.12104961 Pichia pastoris AOX2 AAB57850.1 2104963 Pichia pastoris AOXP04841.1 113652 Hansenula polymorpha AOD1 Q00922.1 231528 Candidaboidinii AOX1 AAQ99151.1 37694459 Ogataea pini

PQQ-Dependent Methanol Dehydrogenase

PQQ-dependent methanol dehydrogenase from M. extorquens (mxaIF) usescytochrome as an electron Gather (Nunn et al, Nucl Acid Res 16:7722(1988)). Methanol dehydrogenase enzymes of methanotrophs such asMethylococcus capsulatis function in a complex with methanemonooxygenase (MMO) (Myronova et al, Biochem 45:11905-14 (2006)). Notethat of accessory proteins, cytochrome CL and PQQ biosynthesis enzymesare needed for active methanol dehydrogenase. Attenuation of one or moreof these required accessory proteins, or retargeting the enzyme to adifferent cellular compartment, would also have the effect ofattenuating PQQ-dependent methanol dehydrogenase activity.

Protein GenBank ID GI Number Organism MCA0299 YP_112833.1 53802410Methylococcus capsulatis MCA0782 YP_113284.1 53804880 Methylococcuscapsulatis mxaI YP_002965443.1 240140963 Methylohacterium extorquensmxaF YP_002965446.1 240140966 Methylobacterium extorquens

DHA Synthase and Other Competing Formaldehyde Assimilation andDissimilation Pathways

Carbon-efficient formaldehyde assimilation can be improved byattenuation of competing formaldehyde assimilation and dissimilationpathways. Exemplary competing assimilation pathways in eukaryoticorganisms include the peroxisomal dissimilation of formaldehyde by DHAsynthase, and the DHA kinase pathway for converting DHA to F6P, bothdescribed herein. Exemplary competing endogenous dissimilation pathwaysinclude one or more of the enzymes shown in FIG. 1.

Methylotrophic yeasts normally target selected methanol assimilation anddissimilation enzymes to peroxisomes during growth on methanol,including methanol oxidase, DHA synthase andS-(hydroxymethyl)-glutathione synthase (see review by Yurimoto et al,supra). The peroxisomal targeting mechanism comprises an interactionbetween the peroxisomal targeting sequence and its correspondingperoxisomal receptor (Lametschwandtner et al, J Biol Chem 273:33635-43(1998)). Peroxisomal methanol pathway enzymes in methylotrophicorganisms contain a PTS1 targeting sequence which binds to a peroxisomalreceptor, such as Pex5p in Candida boidinfi (Horiguchi et al, JBacteriol 183:6372-83 (2001)). Disruption of the PTS1 targetingsequence, the Pex5p receptor and/or genes involved in peroxisomalbiogenesis would enable cytosolic expression of DHA synthase,S-(hydroxymethyl)-glutathione synthase or other methanol-inducibleperoxisomal enzymes. PTS1 targeting sequences of methylotrophic yeastare known in the art (Horiguchi et al, supra). Identification ofperoxisomal targeting sequences of unknown enzymes can be predictedusing bioinformatic methods (eg. Neuberger et al, J Moi Biol 328:581-92(2003))).

Example XIII Methanol Assimilation Via Methanol Dehydrogenase and theRibulose Monophosphate Pathway

This example shows that co-expression of an active methanoldehydrogenase (MeDH) and the enzymes of the Ribulose Monophosphate(RuMP) pathway can effectively assimilate methanol derived carbon.

An experimental system was designed to test the ability of a MeDH inconjunction with the enzymes H6P synthase (HPS) and6-phospho-3-hexuloisomerase (PHI) of the RuMP pathway to assimilatemethanol carbon into the glycolytic pathway and the TCA cycle.Escherichia coli strain ECh-7150 (ΔlacIA, ΔpflB, ΔptsI, ΔPpckA(pckA),ΔPglk(glk), glk::glfB, ΔhycE, ΔfrmR, ΔfrmA, ΔfrmB) was constructed toremove the glutathione-dependent formaldehyde detoxification capabilityencoded by the FrmA and FrmB enzyme. This strain was then transformedwith plasmid pZA23S variants that either contained or lacked gene 2616Aencoding a fusion of the HPS and PHI enzymes. These two transformedstrains were then each transformed with pZS*13S variants that containedgene 2315L (encoding an active MeDH), or gene 2315 RIP2 (encoding acatalytically inactive MeDH), or no gene insertion. Genes 2315 and 2616are internal nomenclatures for NAD-dependent methanol dehydrogenase fromBacillus methanolicus MGA3 and 2616 is a fused phs-hpi constructs asdescribed in Orita et al. (2007) Appl Microbiol Biotechnol 76:439-45.

The six resulting strains were aerobically cultured in quadruplicate, in5 ml minimal medium containing 1% arabinose and 0.6 M 13C-methanol aswell as 100 ug/ml carbenicillin and 25 μg/mlkanamycin to maintainselection of the plasmids, and 1 mM IPTG to induce expression of themethanol dehydrogenase and HPS-PHI fusion enzymes. After 18 hoursincubation at 37° C., the cell density was measuredspectrophotometrically at 600 nM wavelength and a clarified sample ofeach culture medium was submitted for analysis to detect evidence ofincorporation of the labeled methanol carbon into TCA-cycle derivedmetabolites. The label can be further enriched by deleting the gene araDthat competes with ribulose-5-phosphate.

¹³C carbon derived from labeled methanol provided in the experiment wasfound to be significantly enriched in the metabolites pyruvate, lactate,succinate, fumarate, malate, glutamate and citrate, but only in thestrain expressing both catalytically active MeDH 2315L and the HPS-PHIfusion 2616A together (data not shown). Moreover, this strain grewsignificantly better than the strain expressing catalytically activeMeDH but lacking expression of the HPS-PHI fusion (data not shown),suggesting that the HPS-PHI enzyme is capable of reducing growthinhibitory levels of formaldehyde that cannot be detoxified by othermeans in this strain background. These results show that co-expressionof an active MeDH and the enzymes of the RUMP pathway can effectivelyassimilate methanol derived carbon and channel it into TCA-cycle derivedproducts.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties, includingGenBank and GI number publications, are hereby incorporated by referencein this application in order to more fully describe the state of the artto which this invention pertains. Although the invention has beendescribed with reference to the examples provided above, it should beunderstood that various modifications can be made without departing fromthe spirit of the invention.

1. A non-naturally occurring microbial organism having a methanolmetabolic pathway and an acetyl-CoA pathway, wherein said methanolmetabolic pathway comprises 2A or 2J, wherein 2A is a methanolmethyltransferase, wherein 2J is a methanol dehydrogenase, wherein saidacetyl-CoA pathway (3) 1U and 1V; or (4) 1U, 1W, and 1X; wherein 1U is axylulose-5-phosphate phosphoketolase, wherein 1V is aphosphotransacetylase, wherein 1X is an acetyl-CoA transferase, anacetyl-CoA synthetase, or an acetyl-CoA ligase, wherein an enzyme of themethanol metabolic pathway or the acetyl-CoA pathway is encoded by atleast one exogenous nucleic acid and is expressed in a sufficient amountto enhance carbon flux through acetyl-CoA.
 2. The non-naturallyoccurring microbial organism of claim 1, wherein said microbial organismfurther comprises a formaldehyde fixation pathway, wherein saidformaldehyde fixation pathway comprises: (1) 1D and 1Z; (2) 1D; or (3)1B and 1C, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1Cis a 6-phospho-3-hexuloisomerase, wherein 1D is a dihydroxyacetonesynthase, wherein 1Z is a fructose-6-phosphate aldolase.
 3. Thenon-naturally occurring microbial organism of claim 2, wherein an enzymeof the formaldehyde fixation pathway is encoded by at least oneexogenous nucleic acid and is expressed in a sufficient amount toenhance carbon flux through acetyl-CoA.
 4. The non-naturally occurringmicrobial organism of claim 1, wherein said methanol metabolic pathwaycomprises a pathway selected from: (1) 2A and 2B; (2) 2A, 2B and 2C; (3)2J, 2K and 2C; (4) 2J, 2M, and 2N; (5) 2J and 2L; (6) 2J, 2L, and 2G;(7) 2J, 2L, and 2I; (8) 2A, 2B, 2C, 2D, and 2E; (9) 2A, 2B, 2C, 2D, and2F; (10) 2J, 2K, 2C, 2D, and 2E; (11) 2J, 2K, 2C, 2D, and 2F; (12) 2J,2M, 2N, and 2O; (13) 2A, 2B, 2C, 2D, 2E, and 2G; (14) 2A, 2B, 2C, 2D,2F, and 2G; (15) 2J, 2K, 2C, 2D, 2E, and 2G; (16) 2J, 2K, 2C, 2D, 2F,and 2G; (17) 2J, 2M, 2N, 2O, and 2G; (18) 2A, 2B, 2C, 2D, 2E, and 2I;(19) 2A, 2B, 2C, 2D, 2F, and 2I; (20) 2J, 2K, 2C, 2D, 2E, and 2I; (21)2J, 2K, 2C, 2D, 2F, and 2I; and (22) 2J, 2M, 2N, 2O, and 2I, wherein 2Ais a methanol methyltransferase, wherein 2B is amethylenetetrahydrofolate reductase, wherein 2C is amethylenetetrahydrofolate dehydrogenase, wherein 2D is amethenyltetrahydrofolate cyclohydrolase, wherein 2E is aformyltetrahydrofolate deformylase, wherein 2F is aformyltetrahydrofolate synthetase, wherein 2G is a formate hydrogenlyase, wherein 2I is a formate dehydrogenase, wherein 2J is a methanoldehydrogenase, wherein 2K is a formaldehyde activating enzyme orspontaneous, wherein 2L is a formaldehyde dehydrogenase, wherein 2M is aS-(hydroxymethyl)glutathione synthase or spontaneous, wherein 2N is aglutathione-dependent formaldehyde dehydrogenase, wherein 20 is aS-formylglutathione hydrolase,
 5. The non-naturally occurring microbialorganism of claim 4, wherein said microbial organism comprises: (a) one,two, three, four, five, or six exogenous nucleic acids each encoding amethanol metabolic pathway enzyme; or (b) exogenous nucleic acidsencoding each of the enzymes of at least one of the pathways selectedfrom (1)-(22).
 6. (canceled)
 7. The non-naturally occurring microbialorganism of claim 1, wherein said microbial organism comprises: (a) one,two, or three exogenous nucleic acids each encoding an acetyl-CoApathway enzyme; or (b) exogenous nucleic acids encoding each of theenzymes of at least one of the pathways selected from (1)-(4). 8.(canceled)
 9. The non-naturally occurring microbial organism of claim 1,wherein said microbial organism further comprises a formate assimilationpathway, wherein said formate assimilation pathway comprises a pathwayselected from: (1) 1E; (2) 1F, and 1G; (3) 1H, 1I, 1J, and 1K; (4) 1H,1I, 1J, 1L, 1M, and 1N; (5) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (6) 1F, 1G,1H, 1I, 1J, 1L, 1M, and 1N; (7) 1K, 1H, 1I, 1J, 1L, 1M, and 1N; and (8)1H, 1I, 1J, 1O, and 1P, wherein 1E is a formate reductase, 1F is aformate ligase, a formate transferase, or a formate synthetase, wherein1G is a formyl-CoA reductase, wherein 1H is a formyltetrahydrofolatesynthetase, wherein 1I is a methenyltetrahydrofolate cyclohydrolase,wherein 1J is a methylenetetrahydrofolate dehydrogenase, wherein 1K is aformaldehyde-forming enzyme or spontaneous, wherein 1L is a glycinecleavage system, wherein 1M is a serine hydroxymethyltransferase,wherein 1N is a serine deaminase, wherein 1O is amethylenetetrahydrofolate reductase, wherein 1P is an acetyl-CoAsynthase.
 10. The non-naturally occurring microbial organism of claim 9,wherein an enzyme of the formate assimilation pathway is encoded by atleast one exogenous nucleic acid and is expressed in a sufficient amountto enhance carbon flux through acetyl-CoA.
 11. A non-naturally occurringmicrobial organism having a formaldehyde fixation pathway, a formateassimilation pathway and an acetyl-CoA pathway, wherein saidformaldehyde fixation pathway comprises: (1) 1D and 1Z; (2) 1D; or (3)1B and 1C, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1Cis a 6-phospho-3-hexuloisomerase, wherein 1D is a dihydroxyacetonesynthase, wherein 1Z is a fructose-6-phosphate aldolase, wherein saidformate assimilation pathway comprises a pathway selected from: (4) 1E;(5) 1F, and 1G; (6) 1H, 1I, 1J, and 1K; (7) 1H, 1I, 1J, 1L, 1M, and 1N;(8) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (9) 1F, 1G, 1H, 1I, 1J, 1L, 1M, and1N; (10) 1K, 1H, 1I, 1J, 1L, 1M, and 1N; and (11) 1H, 1I, 1J, 1O, and1P, wherein 1E is a formate reductase, 1F is a formate ligase, a formatetransferase, or a formate synthetase, wherein 1G is a formyl-CoAreductase, wherein 1H is a formyltetrahydrofolate synthetase, wherein 1Iis a methenyltetrahydrofolate cyclohydrolase, wherein 1J is amethylenetetrahydrofolate dehydrogenase, wherein 1K is aformaldehyde-forming enzyme or spontaneous, wherein 1L is a glycinecleavage system, wherein 1M is a serine hydroxymethyltransferase,wherein 1N is a serine deaminase, wherein 1O is amethylenetetrahydrofolate reductase, wherein 1P is an acetyl-CoAsynthase, wherein said acetyl-CoA pathway comprises a pathway selectedfrom: (12) 1T and 1V; (13) 1T, 1W, and 1X; (14) 1U and 1V; and (15) 1U,1W, and 1X, wherein 1T is a fructose-6-phosphate phosphoketolase,wherein 1U is a xylulose-5-phosphate phosphoketolase, wherein 1V is aphosphotransacetylase, wherein 1W is an acetate kinase, wherein 1X is anacetyl-CoA transferase, an acetyl-CoA synthetase, or an acetyl-CoAligase, wherein an enzyme of the formaldehyde fixation pathway, theformate assimilation pathway, or the acetyl-CoA pathway is encoded by atleast one exogenous nucleic acid and is expressed in a sufficient amountto enhance carbon flux through acetyl-CoA. 12-23. (canceled)
 24. Thenon-naturally occurring microbial organism of claim 1, wherein saidnon-naturally occurring microbial organism further comprises a pathwaycapable of producing a bioderived compound.
 25. The non-naturallyoccurring microbial organism of claim 24, wherein said bioderivedcompound is an alcohol, a glycol, an organic acid, an alkene, a diene,an organic amine, an organic aldehyde, a vitamin, a nutraceutical or apharmaceutical. 26-29. (canceled)
 30. The non-naturally occurringmicrobial organism of claim 25, wherein said bioderived compounds isselected from the group consisting of: (i) 1,4-butanediol or anintermediate thereto, wherein said intermediate is optionally4-hydroxybutanoic acid (4-HB); (ii) butadiene (1,3-butadiene) or anintermediate thereto, wherein said intermediate is optionally1,4-butanediol, 1,3-butanediol, 2,3-butanediol, crotyl alcohol,3-buten-2-ol (methyl vinyl carbinol) or 3-buten-1-ol; (iii)1,3-butanediol or an intermediate thereto, wherein said intermediate isoptionally 3-hydroxybutyrate (3-HB), 2,4-pentadienoate, crotyl alcoholor 3-buten-1-ol; (iv) adipate, 6-aminocaproic acid, caprolactam,hexamethylenediamine, levulinic acid or an intermediate thereto, whereinsaid intermediate is optionally adipyl-CoA or 4-aminobutyryl-CoA; (v)methacrylic acid or an ester thereof, 3-hydroxyisobutyrate,2-hydroxyisobutyrate, or an intermediate thereto, wherein said ester isoptionally methyl methacrylate or poly(methyl methacrylate); (vi)1,2-propanediol (propylene glycol), 1,3-propanediol, glycerol, ethyleneglycol, diethylene glycol, triethylene glycol, dipropylene glycol,tripropylene glycol, neopentyl glycol, bisphenol A or an intermediatethereto; (vii) succinic acid or an intermediate thereto; and (viii) afatty alcohol, a fatty aldehyde or a fatty acid comprising C4 to C27carbon atoms, C8 to C18 carbon atoms, C12 to C18 carbon atoms, or C12 toC14 carbon atoms, wherein said fatty alcohol is optionally dodecanol(C12; lauryl alcohol), tridecyl alcohol (C13; 1-tridecanol, tridecanol,isotridecanol), myristyl alcohol (C14; 1-tetradecanol), pentadecylalcohol (C15; 1-pentadecanol, pentadecanol), cetyl alcohol (C16;1-hexadecanol), heptadecyl alcohol (C17; 1-n-heptadecanol, heptadecanol)and stearyl alcohol (C18; 1-octadecanol) or palmitoleyl alcohol (C16unsaturated; cis-9-hexadecen-1-ol). 31-42. (canceled)
 43. Thenon-naturally occurring microbial organism of claim 1, wherein saidmicrobial organism is a species of bacteria, yeast, or fungus.
 44. Amethod for producing a bioderived compound, comprising culturing thenon-naturally occurring microbial organism of any claim 24 underconditions and for a sufficient period of time to produce saidbioderived compound.
 45. The method of claim 44, wherein said methodfurther comprises separating the bioderived compound from othercomponents in the culture.
 46. The method of claim 45, wherein theseparating comprises extraction, continuous liquid-liquid extraction,pervaporation, membrane filtration, membrane separation, reverseosmosis, electrodialysis, distillation, crystallization, centrifugation,extractive filtration, ion exchange chromatography, absorptionchromatography, or ultrafiltration. 47-59. (canceled)
 60. Thenon-naturally occurring microbial organism of claim 1, wherein saidacetyl-CoA pathway comprises 1U and 1V 1T and 1V.
 61. The non-naturallyoccurring microbial organism of claim 60, wherein said formaldehydefixation pathway comprises 1D and 1Z.
 62. The non-naturally occurringmicrobial organism of claim 60, wherein said formaldehyde fixationpathway comprises 1B and 1C.
 63. The non-naturally occurring microbialorganism of claim 1, wherein said microbial organism further comprises:(a) attenuation of one or more endogenous enzymes selected from DHAkinase, methanol oxidase, PQQ-dependent methanol dehydrogenase, DHAsynthase or any combination thereof; or (b) a gene disruption of one ormore endogenous nucleic acids encoding enzymes selected from DHA kinase,methanol oxidase, PQQ-dependent methanol dehydrogenase, DHA synthase orany combination thereof. 64-88. (canceled)